Immunogenic composition

ABSTRACT

The present invention relates to immunogenic compositions comprising one or more  Streptococcus pneumoniae  capsular saccharide conjugates and a protein component comprising Protein E and/or PilA from  Haemophilus influenzae.

This application is a United States 111(a) application which claimspriority from Provisional Application Serial Number 1218660.7, filed inthe United Kingdom Oct. 17, 2012, the contents of which is hereinincorporated by reference in its entirety.

SEQUENCE LISTING

This application contains sequences, listed in an electronic SequenceListing entitled VB65265T_Seq_List, 258 KB, created on Nov. 12, 2013,the contents and sequences of which are hereby incorporated by referenceherein.

TECHNICAL FIELD

The present invention relates to immunogenic compositions comprising oneor more Streptococcus pneumoniae capsular saccharide conjugates and aprotein component comprising Protein E and/or PilA from Haemophilusinfluenzae.

BACKGROUND

Non-typeable Haemophilus influenzae (NTHi) is an important and commonrespiratory pathogen that causes otitis media in infants and children.NTHi is, after Streptococcus pneumoniae, the most common cause of acuteotitis media in children (J. Immunology 183: 2593-2601 (2009),Pediatrics 113:1451-1465 (2004)). It is an important cause of sinusitisin children and adults. (Current Infectious Disease Reports 11:177-182(2009)). It has been associated with increased risk of exacerbations inchronic obstructive pulmonary disease (COPD) in adults. (Journal ofChronic Obstructive Pulmonary Disease 3:109-115 (2006)). In addition,non-typeable H. influenzae causes community-acquired pneumonia in adultsand may cause pneumonia in children in developing countries. (CurrentInfectious Disease Reports 11:177-182 (2009)).

Streptococcus pneumoniae (S. pneumoniae), also known as thepneumococcus, is a Gram-positive bacterium. S. pneumoniae is a majorpublic health problem all over the world and is responsible forconsiderable morbidity and mortality, especially among infants, theelderly and immunocompromised persons. S. pneumoniae causes a wide rangeof important human pathologies including community-acquired pneumonia,acute sinusitis, otitis media, meningitis, bacteremia, septicemia,osteomyelitis, septic arthritis, endocarditis, peritonitis,pericarditis, cellulitis, and brain abscess. S. pneumoniae is estimatedto be the causal agent in 3,000 cases of meningitis, 50,000 cases ofbacteremia, 500,000 cases of pneumonia, and 7,000,000 cases of otitismedia annnually in the United States alone (Reichler, M. R. et al.,1992, J. Infect. Dis. 166: 1346; Stool, S. E. and Field, M. J., 1989Pediatr. Infect. Dis J. 8: S11). Mortality rates due to pneumococcaldisease are especially high in children younger than 5 years of age fromboth developed and developing countries. The elderly, theimmunocompromised and patients with other underlying conditions(diabetes, asthma) are also particularly susceptible to disease.

The major clinical syndromes caused by S. pneumoniae are widelyrecognized and discussed in standard medical textbooks (Fedson D S,Muscher D M. In: Plotkin S A, Orenstein W A, editors. Vaccines. 4thedition. Philadelphia WB Saunders Co, 2004a: 529-588). For instance,Invasive pneumococcal disease (IPD) is defined as any infection in whichS. pneumoniae is isolated from the blood or another normally sterilesite (Musher D M. Streptococcus pneumoniae. In Mandell G L, Bennett J E,Dolin R (eds). Principles and Practice of Infectious diseases (5th ed).New York, Churchill Livingstone, 2001, p 2128-2147).

Chronic obstructive pulmonary disease is a chronic inflammatory diseaseof the lungs and a major cause of morbidity and mortality worldwide.Approximately one in 20 deaths in 2005 in the US had COPD as theunderlying cause. (Drugs and Aging 26:985-999 (2009)). It is projectedthat in 2020 COPD will rise to the fifth leading cause of disabilityadjusted life years, chronic invalidating diseases, and to the thirdmost important cause of mortality (Lancet 349:1498-1504 (1997)).

Thus a need for combination vaccines against Streptococcus pneumoniaeand Haemophilus influenzae exists.

Protein E (PE) is an outer membrane lipoprotein with adhesiveproperties. It plays a role in the adhesion/invasion of non-typeableHaemophilus influenzae (NTHi) to epithelial cells. (J. Immunology 183:2593-2601 (2009); The Journal of Infectious Diseases 199:522-531 (2009),Microbes and Infection 10:87-96 (2008)). It is highly conserved in bothencapsulated Haemophilus influenzae and non-typeable H. influenzae andhas a conserved epithelial binding domain. (The Journal of InfectiousDiseases 201:414-419 (2010)). Thirteen different point mutations havebeen described in different Haemophilus species when compared withHaemophilus influenzae Rd as a reference strain. Its expression isobserved on both logarithmic growing and stationary phase bacteria.(WO2007/084053).

Protein E is also involved in human complement resistance throughbinding vitronectin. (Immunology 183: 2593-2601 (2009)). PE, by thebinding domain PKRYARSVRQ YKILNCANYH LTQVR (SEQ ID NO. 1, correspondingto amino acids 84-108 of SEQ ID NO. 4), binds vitronectin which is animportant inhibitor of the terminal complement pathway. (J. Immunology183:2593-2601 (2009)).

Pilin A (PilA) is likely the major pilin subunit of H. influenzae TypeIV Pilus (Tfp) involved in twitching motility (Infection and Immunity,73: 1635-1643 (2005)). NTHi PilA is a conserved adhesin expressed invivo. It has been shown to be involved in NTHi adherence, colonizationand biofilm formation. (Molecular Microbiology 65: 1288-1299 (2007)).

The inventors have found that PilA and PE may be beneficially present inan immunogenic composition for prevention of H. influenzae andfurthermore that PilA and Protein E can be added to a compositioncomprising S. pneumoniae capsular saccharides in order to provide animmunogenic composition which can prevent H. influenzae and S.pneumoniae infection. The skilled person would be aware of the effect ofcarrier-induced epitopic suppression, and know that generallysimultaneous exposure to multiple conjugate antigens can result ineither enhanced or diminished immune responses (Plotkin et al, Vaccinesfourth addition 2003).

BRIEF SUMMARY

The inventors have found that PilA, PE (or fragments thereof) andsaccharides from Streptococcus pneumoniae may be beneficially combinedin an immunogenic composition to provide effective protection against H.influenzae and S. pneumoniae.

Accordingly in a first aspect there is provided an immunogeniccomposition comprising 1 or more (e.g. 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20) Streptococcus pneumoniae capsular saccharideconjugates and a protein component comprising Protein E or animmunogenic fragment of Protein E and/or PilA (or an immunogenicfragment of PilA) from Haemophilus influenzae.

In a second aspect there is provided a vaccine comprising an immunogeniccomposition of the invention and a pharmaceutically acceptableexcipient.

In a third aspect there is provided a method of immunising a subjectagainst diseases caused by Streptococcus pneumoniae infection comprisingadministering to the subject a therapeutically effective dose of theimmunogenic composition of the invention or the vaccine of theinvention.

In a fourth aspect there is provided a method of immunising a subjectagainst diseases caused by Haemophilus influenzae infection comprisingadministering to the subject a therapeutically effective dose of theimmunogenic composition of the invention or the vaccine of theinvention.

In a fifth aspect there is provided an immunogenic composition of theinvention or a vaccine of the invention for use in the treatment orprevention of diseases caused by Streptococcus pneumoniae infection.

In a sixth aspect there is provided an immunogenic composition of theinvention or a vaccine of the invention for use in the treatment orprevention of diseases caused by Haemophilus influenzae infection.

In a seventh aspect there is provided a use of the immunogeniccomposition of the invention or the vaccine of the invention in themanufacture of a medicament for the treatment or prevention of diseasescaused by Streptococcus pneumoniae infection.

In an eighth aspect there is provided a use of the immunogeniccomposition of the invention or the vaccine of the invention in themanufacture of a medicament for the treatment or prevention of diseasescaused by Haemophilus influenzae infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. SDS-PAGE of induced bacterial extracts for fusion proteinconstructs LVL291, LVL268 and LVL269. Insoluble fraction (I), Solublefraction (S) and Culture Media fraction (M) were loaded for LVL291,LVL268 and LVL269 before and after induction (ind).

FIG. 2. SDS-PAGE and Western blot related to purification extracts forfusion protein constructs LVL291, LVL268 and LVL269. Flow throughfraction (Ft), Wash fraction (W) and Elution fraction (E) were loadedfor purification of LVL291, LVL268 and LVL269. Anti-his tag was used toprobe extracts.

FIG. 3. SDS-PAGE of induced bacterial and purification extracts forfusion protein constructs LVL291 and LVL315. Culture Media fraction (M),Soluble fraction (Sol), Insoluble fraction (Ins), Flow through fraction(Ft), Wash fraction #1 (W1), Wash fraction #2 (W2) and Elution fraction(E) were loaded for LVL291 and LVL315.

FIG. 4. SDS-PAGE of induced bacterial and purification extracts forfusion protein construct LVL312. Culture Media fraction (M), Solublefraction (Sol), Insoluble fraction (Ins), Flow Through fraction (Ft),Wash fraction #1 (W1), Wash fraction #2 (W2) and Elution fraction (E)were loaded for LVL312.

FIG. 5. SDS-PAGE of induced (1 mM and 10 μM IPTG) bacterial extracts forfusion protein construct LVL317. Extracts from before (NI) and afterinduction (In), Soluble fraction (S), Insoluble fraction (I).

FIG. 6. SDS-PAGE of induced (1 mM and 10 μM IPTG) bacterial extracts forfusion protein construct LVL318. Extracts from before (NI) and afterinduction (In), Culture Media fraction (M), Soluble fraction (S),Insoluble fraction (I).

FIG. 7. CD spectra of PE, PilA and PE-PilA fusion proteins.

FIG. 8. Combination of PE and PilA CD spectrum.

FIG. 9. PilA thermal denaturation curve.

FIG. 10. PE denaturation curve.

FIG. 11. PE-PilA fusion protein thermal denaturation curve.

FIG. 12. Typical SP Sepharose™ Fast Flow chromatogram.

FIG. 13. Typical Q Sepharose™ Fast Flow chromatogram.

FIG. 14. SDS-PAGE of In-process samples from purification process ofPE-PilA fusion protein.

FIG. 15. Western Blot of In-process samples of purification process fromPE-PilA fusion protein. Blot using rabbit polyclonal anti-PE.

FIG. 16. Western Blot of In-process samples of purification process fromPE-PilA fusion protein. Blot using rabbit polyclonal anti-E. coli (BLR).

FIG. 17. Thermal transition of PE-PilA fusion protein and PE and PilAproteins. Curves: PilA (1), Protein E (Prot E, PE) (2), PE-PilA PurifiedBulk not diluted, 737 μg/ml (3), and PE-PilA Purified Bulk diluted atFinal Container concentration 60 μg/ml (4).

FIG. 18. Antibody responses against LVL291 PE-PilA fusion protein andagainst monovalent PE and PilA in the Balb/c mouse model.

FIG. 19. Effect of PE-PilA fusion protein vaccination on NTHi strain86-028NP bacterial clearance in mouse nasopharynx.

FIG. 20. Effect of PE-PilA fusion protein vaccination on NTHi strain3224A bacterial clearance in mouse nasopharynx.

FIG. 21. Effect of PilA vaccination on bacterial clearance in mousenasopharynx.

FIG. 22. Effect of PE vaccination on bacterial clearance in mousenasopharynx.

FIG. 23. (a) LVL317 PE-PilA fusion protein binding to vitronectin and(b) LVL317 and LVL735 PE-PilA fusion protein bound to vitronectin.

FIG. 24. Inhibition of vitronectin binding by polyclonal antibodiesagainst PE-PilA fusion protein.

FIG. 25. SDS-PAGE of soluble fractions of induced bacterial extracts forfusion protein constructs LVL291, LVL702, LVL736, LVL737, LVL738,LVL739, LVL740 and pET26b vector (negative control). (a) Experiment 1(b) Experiment 2 (c) Experiment 3. PE-PilA fusion protein indicated byarrow.

FIG. 26. The average band percentage of fusion protein in the solublefraction from Experiments 1, 2 and 3.

FIG. 27. PE and PilA antibody response to LVL317 and LVL735.

FIG. 28. Effect of LVL735 and LVL317 vaccination on bacterial clearancein a mouse model of non-typeable Haemophilus influenzae nasopharyngealcolonization.

FIG. 29. Graph comparing the immunogenicity of a composition comprising12 saccharide conjugates, PhtD, dPly and PE-PilA (12V+prot) with acomposition comprising 12 saccharide conjugates (12V) and a compositioncomprising 10 saccharide conjugates (10V) as measured after injection ofmice using an anti-saccharide ELISA assay. GMC=geometric meanconcentration. IC=confidence intervals.

FIG. 30. Graph comparing the immunogenicity of a composition comprising12 saccharide conjugates, PhtD, dPly and PE-PilA (12V+prot) with acomposition comprising 12 saccharide conjugates (12V) and a compositioncomprising 10 saccharide conjugates (10V) as measured after injection ofmice using an opsonophagocytosis assay. GMT=geometric means titer.

FIG. 31. Graph comparing the immunogenicity of a composition comprising12 saccharide conjugates, PhtD, dPly and PE-PilA (12V+prot) with acomposition comprising PhtD, dPly and PE-PilA alone (prot) as measuredafter injection of mice using an anti-protein ELISA assay. GMC=geometricmean concentration. IC=confidence intervals.

FIG. 32. Graph comparing the immunogenicity of a composition comprising12 saccharide conjugates, PhtD, dPly and PE-PilA (12V+prot) with acomposition comprising 12 saccharide conjugates (12V) and a compositioncomprising 10 saccharide conjugates (10V) as measured after injection ofguinea pigs using an opsonophagocytosis assay. GMT=geometric meanstiter.

FIG. 33. Graph comparing the immunogenic of a composition comprising 12saccharide conjugates, PhtD, dPly and PE-PilA (12V+prot) with acomposition comprising 12 saccharide conjugates (12V) and a compositioncomprising 10 saccharide conjugates (10V) as measured after injection ofguinea pigs using an anti-saccharide ELISA. GMC=geometric meanconcentration. IC=confidence intervals.

FIG. 34. Graph comparing the immunogenic of a composition comprising 12saccharide conjugates, PhtD, dPly and PE-PilA (12V+prot) with acomposition comprising PhtD, dPly and PE-PilA alone (prot) as measuredafter injection of guinea pigs using an anti-protein ELISA.GMC=geometric mean concentration. ic=confidence intervals.

DETAILED DESCRIPTION

In a first aspect, the present invention relates to an immunogeniccomposition comprising 1 or more (e.g. 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20) Streptococcus pneumoniae capsular saccharideconjugates and a protein component comprising Protein E (or animmunogenic fragment thereof) and/or PilA (or an immunogenic fragmentthereof) from Haemophilus influenzae.

The term “protein component” relates to a sequence of amino acidscomprising Protein E (or an immunogenic fragment thereof) and/or PilA(or an immunogenic fragment thereof), the protein component may compriseProtein E alone, PilA alone, an immunogenic fragment of Protein E alone,an immunogenic fragment of PilA alone, Protein E and PilA, animmunogenic fragment of Protein E and PilA, an immunogenic fragment ofProtein E and an immunogenic fragment of PilA or Protein E and animmunogenic fragment of PilA (for example as a fusion protein). Theprotein component may further comprise additional sequences.

Protein E

As used herein “Protein E”, “protein E”, “Prot E”, and “PE” mean ProteinE from H. influenzae. Protein E may consist of or comprise the aminoacid sequence of SEQ ID NO. 4 (MKKIILTLSL GLLTACSAQI QKAEQNDVKLAPPTDVRSGY IRLVKNVNYY IDSESIWVDN QEPQIVHFDA VVNLDKGLYV YPEPKRYARSVRQYKILNCA NYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICANYGEAFSVDKK) as well as sequences with at least or exactly 75%, 77%, 80%,85%, 90%, 95%, 97%, 99% or 100% identity, over the entire length, to SEQID NO. 4. Comparison of 53 sequences of Protein E from Haemophilusinfluenzae (Table 1, SEQ ID NO. 5-SEQ ID NO. 57) demonstratedapproximately 77% to approximately 100% identity to Protein E as setforth in SEQ ID NO. 4. For example, in the amino acid sequence ofProtein E, amino acid #20 may be isoleucine (I) or threonine (T); aminoacid #23 may be alanine (A) or valine (V); amino acid #24 may be lysine(K) or glutamic acid (E); amino acid #31 may be alanine (A) or threonine(T); amino acid #32 may be proline (P) or alanine (A); amino acid #34may be threonine (T) or alanine (A); amino acid #37 may be arginine (R)or glutamine (Q); amino acid #47 may be valine (V) or alanine (A); aminoacid #57 may be tryptophane (W) or may be absent (−); amino acid #70 maybe alanine (A) or threonine (T); amino acid #93 may be glutamine (Q) orabsent (−); amino acid #109 may be threonine (T) or isoleucine (I);amino acid #119 may be glycine (G) or serine (S); amino acid #153 may beglutamic acid (E) or lysine (K); amino acid #156 may be serine (S) orleucine (L); amino acid #160 may be lysine (K) or asparagine (N); aminoacid #161 may be lysine (K), isoleucine (I) or absent (−); amino acids#162-#195 may be absent, or as set forth in SEQ ID NO. 15 (with (−)indicating amino acid #166 is absent) or as set forth in SEQ ID NO. 16;or any combination thereof.

Protein E may consist of or comprise an amino acid sequence that differsfrom SEQ ID NO. 4 at any one or more amino acid selected from the groupconsisting of: amino acid #20, amino acid #23, amino acid #24, aminoacid #31, amino acid #32, amino acid #34, amino acid #37, amino acid#47, amino acid #57, amino acid #70, amino acid #93, amino acid #109,amino acid #119, amino acid #153, amino acid #156, amino acid #160,amino acid #161 and amino acids #162-#195, wherein amino acid #20 isthreonine (T); amino acid #23 is valine (V); amino acid #24 is lysine(K); amino acid #31 is threonine (T); amino acid #32 is alanine (A);amino acid #34 is alanine (A); amino acid #37 is glutamine (Q); aminoacid #47 is alanine (A); amino acid #57 is absent (−); amino acid #70 isthreonine (T); amino acid #93 is absent (−); amino acid #109 isisoleucine (I); amino acid #119 is serine (S); amino acid #153 is lysine(K); amino acid #156 is leucine (L); amino acid #160 is asparagine (N);amino acid #161 is lysine (K) or isoleucine (I); or amino acids#162-#195 are as set forth in SEQ ID NO. 15 (with (−) indicating aminoacid #166 is absent) or as set forth in SEQ ID NO. 16.

TABLE 1Protein E amino acid sequences from 53 strains of Haemophilus influenzae(SEQ ID NO. 5 - SEQ ID NO. 57). Strain Name Protein E sequence 3224AMKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDK K (SEQ ID NO. 5)RdKW20 MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDRGLYVYPEPKRYARSVRQYKILNCANYHLTQIRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 6)86-028NP MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDK K (SEQ ID NO. 7)R2846 MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDK K (SEQ ID NO. 8)R2866 MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDK K (SEQ ID NO. 9)3655 MKKIILTLSLGLLTACSAQIQKAEQNDMKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVD KK (SEQ ID NO. 10)PittAA MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDK K (SEQ ID NO. 11)PittEE MKKIILTLSLGLLTACSAQIQKAEQNDMKLAPPTDVRSGYIRLVKNVNYYIDSE SI-VDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK(SEQ ID NO. 12) PittHHMKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDTVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDK K (SEQ ID NO. 13)PittII MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDK K (SEQ ID NO. 14)R3021 MKKIILTLSLGLLTACSAQTQKAEQNDVKLTPPTDVQSGYVRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRIDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKNKKICT-LISLNFIQLLGCREYSIFLQLLLFYC WHF (SEQ ID NO. 15) 22.4-21MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKKIKKICTLISLNFIQLLGCREYSIFLQLLLFYCWHF (SEQ ID NO. 16) 3219CMKKIILTLSLGLLTACSAQIQKAEQNDMKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVD KK (SEQ ID NO. 17)3185 MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDK K (SEQ ID NO. 18)3241A MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDK K (SEQ ID NO. 19)038144S1 MKKIILTLSLGLLTACSAQTQKVEQNDVKLTAPTDVRSGFVRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFLVDK K (SEQ ID NO. 20)810956 MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDK K (SEQ ID NO. 21)821246 MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQIRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 22)840645 MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDK K (SEQ ID NO. 23)902550Z19 MKKIILTLSLGLLTACSAQTQKVEQNDVKLTPPTDVRSGYVRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVD KK (SEQ ID NO. 24)A840177 MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDK K (SEQ ID NO. 25)A860514 MKKIILTLSLGLLTACSAQTQKVEQNDVKLTAPTDVRSGYVRLVKNANYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVD KK (SEQ ID NO. 26)A950014 MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRIDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 27)306543X4 MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDK K(SEQ ID NO. 28)A930105 MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDTVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDK K(SEQ ID NO. 29)901905U MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDK K (SEQ ID NO. 30)A920030 MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDK K (SEQ ID NO. 31)32216 MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDK K (SEQ ID NO. 32)27W116791N MKKIILTLSLGLLTACSAQTQKVEQNDVKLTPPTDVRSGYVRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVD KK (SEQ ID NO. 33)N218 MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDK K (SEQ ID NO. 34)N163 MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDK K (SEQ ID NO. 35)N162 MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDK K (SEQ ID NO. 36)N107 MKKIILTLSLGLLTACSAQTQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQIRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDK K (SEQ ID NO. 37)N91 MKKIILTLSLGLLTACSAQTQKVEQNDVKLTAPADVRSGYVRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVD KK (SEQ ID NO. 38)D211PG MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVR-YKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDKK (SEQ ID NO. 39) D211PDMKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVR-YKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDKK (SEQ ID NO. 40) D201PGMKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDK K (SEQ ID NO. 41)D201PD MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDK K (SEQ ID NO. 42)D198PG MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDK K (SEQ ID NO. 43)D198PD MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDK K (SEQ ID NO. 44)D195PD MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDTVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQSLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 45)D189PG MKKIILTLSLGLLTACSAQTQKVEQNDVKLTPPTDVRSGYVRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTVYNAAQIICANYGKAFSVD KK (SEQ ID NO. 46)D189PD MKKIILTLSLGLLTACSAQTQKVEQNDVKLTPPTDVRSGYVRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTVYNAAQIICANYGKAFSVD KK (SEQ ID NO. 47)D129CG MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDK K (SEQ ID NO. 48)D124PG MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDTVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDK K (SEQ ID NO. 49)D124PD MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDTVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDK K(SEQ ID NO. 50)D58PG MKKIILTLSLGLLTACSAQTQKAEQNDVKLTPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVD KK (SEQ ID NO. 51)D33OD MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDK K (SEQ ID NO. 52)BS433 MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDTVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDK K (SEQ ID NO. 53)BS432 MKKIILTLSLGLLTACSAQTQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQIRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDK K(SEQ ID NO. 54)1714 MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDK K (SEQ ID NO. 55)1128 MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDK K (SEQ ID NO. 56)BS430 MKKIILTLSLGLLTACSAQIQKAEQNDMKLAPPTDVRSGYIRLVKNVNYYIDSE SI-VDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 57) - indicatesamino acid is absent.

Protein E may be Protein E from H. influenzae strain 3224A, RdKW20,86-028NP, R2846, R2866, 3655, PittAA, PittEE, PittHH, PittII, R3021,22.4-21, 3219C, 3185, 3241A, 038144S1, 810956, 821246, 840645,902550Z19, A840177, A860514, A950014, 306543X4, A930105, 901905U,A920030, 3221B, 27W116791N, N218, N163, N162, N107, N91, D211PG, D211PD,D201PG, D201PD, D198PG, D198PD, D195PD, D189PG, D189PD, D129CG, D124PG,D124PD, D58PG, D330D, BS433, BS432, 1714, 1128 or BS430. Protein E maybe Protein E as set forth in any of SEQ ID NO. 5-SEQ ID NO. 57.

Protein E may be a sequence with at least 95% or 98%, 99% identity, overthe entire length, to any of SEQ ID NO. 4-SEQ ID NO. 57. Protein E maybe a sequence with at least 95% identity, over the entire length, to anyof the sequences set forth in Table 1, SEQ ID NO. 5-SEQ ID NO. 57.

Immunogenic fragments of Protein E comprise immunogenic fragments of atleast 7, 10, 15, 20, 25, 30 or 50 contiguous amino acids of SEQ ID NO.4. In one embodiment the fragment is less than 150, 125, 100, 75, or 60amino acids of Protein E, for example in one embodiment the immunogeniccomposition of the invention comprises less than 150, 125, 100, 75 or 60amino acids of Protein E. The immunogenic fragments may elicitantibodies which can bind SEQ ID NO. 4. The immunogenic fragment maycomprise a B and/or T cell epitope of SEQ ID NO:4.

Immunogenic fragments of Protein E may comprise immunogenic fragments ofat least 7, 10, 15, 20, 25, 30 or 50 contiguous amino acids of any ofSEQ ID NO. 4-SEQ ID NO. 57. The immunogenic fragments may elicitantibodies which can bind the full length sequence from which thefragment is derived. The immunogenic fragment may comprise a B and/or Tcell epitope of SEQ ID NO:4-SEQ ID NO. 57. In one embodiment theimmunogenic fragment of Protein E is selected from the group consistingof amino acids 17-160 of SEQ ID NO. 4 (SEQ ID NO. 122), amino acids18-160 of SEQ ID NO. 4 (SEQ ID NO. 123), amino acids 19-160 of SEQ IDNO. 4 (SEQ ID NO. 124), amino acids 20-160 of SEQ ID NO. 4 (SEQ ID NO.125) and amino acids 22-160 of SEQ ID NO. 4 (SEQ ID NO. 126). In anotherembodiment, the immunogenic fragment of protein E is selected from thegroup consisting of amino acids 17-160 of SEQ ID NO. 4 (SEQ ID NO. 122),amino acids 18-160 of SEQ ID NO. 4 (SEQ ID NO. 123), amino acids 19-160of SEQ ID NO. 4 (SEQ ID NO. 124), amino acids 20-160 of SEQ ID NO. 4(SEQ ID NO. 125), amino acids 22-160 of SEQ ID NO. 4 (SEQ ID NO. 126),amino acids 23-160 of SEQ ID NO. 4 (SEQ ID NO. 179) and amino acids24-160 of SEQ ID NO. 4 (SEQ ID NO. 180). In a further embodiment, theimmunogenic fragment of Protein E from H. influenzae is selected fromthe group consisting of amino acids 17-160 of SEQ ID NO. 4 (SEQ ID NO.122), amino acids 18-160 of SEQ ID NO. 4 (SEQ ID NO. 123), amino acids20-160 of SEQ ID NO. 4 (SEQ ID NO. 125), amino acids 22-160 of SEQ IDNO. 4 (SEQ ID NO. 126), amino acids 23-160 of SEQ ID NO. 4 (SEQ ID NO.179) and amino acids 24-160 of SEQ ID NO. 4 (SEQ ID NO. 180). Morespecifically, in one embodiment, the immunogenic fragment is SEQ ID NO.124, amino acids 19-160 of SEQ ID NO. 4. In an additional embodiment,the immunogenic fragment is SEQ ID NO. 125, amino acids 20-160 of SEQ IDNO. 5. In another embodiment, the immunogenic fragment is an immunogenicfragment of Protein E from H. influenzae selected from the groupconsisting of amino acids 23-160 of SEQ ID NO. 4 (SEQ ID NO. 179) andamino acids 24-160 of SEQ ID NO. 4 (SEQ ID NO. 180).

Protein E contains an epithelial cell binding region (PKRYARSVRQYKILNCANYH LTQVR, SEQ ID NO. 128) that has been reported to be conservedamong more than 100 clinical NTHi isolates, encapsulated H. influenzae,and culture collection strains analyzed (Singh et al, J. Infect. Dis.201(3):414-9 (2010)). Singh et al. reported that Protein E was highlyconserved in both NTHi and encapsulated H. influenzae (96.9%-100%identity without the signal peptide). In one embodiment, the fragment ofProtein E comprises the binding region of SEQ ID NO. 128 (PKRYARSVRQYKILNCANYH LTQVR)

Protein E - SEQ ID NO. 4MKKIILTLSL GLLTACSAQI QKAEQNDVKL APPTDVRSGY IRLVKNVNYY IDSESIWVDNQEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCA NYHLTQVRTD FYDEFWGQGLRAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKKAmino acids 17-160 of Protein E from SEQ ID NO. 4 - SEQ ID NO. 122      SAQI QKAEQNDVKL APPTDVRSGY IRLVKNVNYY IDSESIWVDN QEPQIVHFDAVVNLDKGLYV YPEPKRYARS VRQYKILNCA NYHLTQVRTD FYDEFWGQGL RAAPKKQKKHTLSLTPDTTL YNAAQIICAN YGEAFSVDKKAmino acids 18-160 of Protein E from SEQ ID NO. 4 - SEQ ID NO. 123       AQI QKAEQNDVKL APPTDVRSGY IRLVKNVNYY IDSESIWVDN QEPQIVHFDAVVNLDKGLYV YPEPKRYARS VRQYKILNCA NYHLTQVRTD FYDEFWGQGL RAAPKKQKKHTLSLTPDTTL YNAAQIICAN YGEAFSVDKKAmino acids 19-160 of Protein E from SEQ ID NO. 4 - SEQ ID NO. 124        QI QKAEQNDVKL APPTDVRSGY IRLVKNVNYY IDSESIWVDN QEPQIVHFDAVVNLDKGLYV YPEPKRYARS VRQYKILNCA NYHLTQVRTD FYDEFWGQGL RAAPKKQKKHTLSLTPDTTL YNAAQIICAN YGEAFSVDKKAmino acids 20-160 of Protein E from SEQ ID NO. 4 - SEQ ID NO. 125        I QKAEQNDVKL APPTDVRSGY IRLVKNVNYY IDSESIWVDN QEPQIVHFDAVVNLDKGLYV YPEPKRYARS VRQYKILNCA NYHLTQVRTD FYDEFWGQGL RAAPKKQKKHTLSLTPDTTL YNAAQIICAN YGEAFSVDKKAmino acids 22-160 of Protein E from SEQ ID NO. 4 - SEQ ID NO. 126        KAEQNDVKL APPTDVRSGY IRLVKNVNYYIDSESIWVDN QEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCA NYHLTQVRTDFYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKKAmino acids 23-160 of Protein E from SEQ ID NO. 4 - SEQ ID NO. 179AEQNDVKL APPTDVRSGY IRLVKNVNYYIDSESIWVDN QEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCA NYHLTQVRTDFYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKKAmino acids 24-160 Protein E from SEQ ID NO. 4 - SEQ ID NO. 180           EQNDVKL APPTDVRSGY IRLVKNVNYYIDSESIWVDN QEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCA NYHLTQVRTDFYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKK

In one embodiment the Protein E or an immunogenic fragment thereof iscapable of eliciting an immune response which recognizes SEQ ID NO:4.Whether or not a first protein is capable of eliciting an immuneresponse which recognizes a second protein can be determined using anELISA assay (for example the ELISA described in example 22).

PilA

As used herein “PilA” means Pilin A from H. influenzae. PilA may consistof or comprise the protein sequence of SEQ ID NO. 58 (MKLTTQQTLKKGFTLIELMI VIAIIAILAT IAIPSYQNYT KKAAVSELLQ ASAPYKADVE LCVYSTNETTNCTGGKNGIA ADITTAKGYV KSVTTSNGAI TVKGDGTLAN MEYILQATGN AATGVTWTTTCKGTDASLFP ANFCGSVTQ) as well as sequences with 80% to 100% identity toSEQ ID NO. 58. For example, PilA may be at least 80%, 85%, 90%, 95%, 97%or 100% identical to SEQ ID NO. 58. Full length comparison of 64sequences of PilA from Haemophilus influenzae (Table 2, SEQ ID NO.58-SEQ ID NO. 121) demonstrated approximately 80% to 100% identity toPilA as set forth in SEQ ID NO. 58. For example, in the amino acidsequence of PilA, amino acid #6 may be glutamine (Q) or leucine (L);amino acid #7 may be glutamine (Q) or threonine (T); amino acid #37 maybe glutamine (Q) or lysine (K); amino acid #44 may be alanine (A) orserine (S); amino acid #57 may be alanine (A) or serine (S); amino acid#67 may be asparagine (N) or glycine (G); amino acid #68 may be glutamicacid (E) or lysine (K); amino acid #69 may be theronine (T) or proline(P); amino acid #71 may be lysine (K), asparagine (N), serine (S) orthreonine (T); amino acid #73 may be threonine (T), serine (S) ormethionine (M); amino acid #76 may be lysine (K), serine (S) orasparagine (N); amino acid #84 may be threonine (T) or lysine (K); aminoacid #86 may be alanine (A) or valine (V); amino acid #91 may be lysine(K) or alanine (A); amino acid #94 may be threonine (T), isoleucine (I)or lysine (K); amino acid #96 may be serine (S) or glutamine (Q); aminoacid #97 may be asparagine (N) or serine (S); amino acid #99 may bealanine (A) or glycine (G); amino acid #103 may be alanine (A) or lysine(K); amino acid #109 may be aspartic acid (D), alanine (A) or threonine(T); amino acid #110 may be glycine (G), asparagine (N), or arginine(R); amino acid #112 may be serine (S) or glutamic acid (E); amino acid#114 may be threonine (T) or isoleucine (I); amino acid #116 may bethreonine (T) or glutamine (Q); amino acid #118 may be glutamic acid(E), threonine (T), alanine (A), lysine (K) or serine (S); amino acid#121 may be serine (S) or alanine (A); amino acid #122 may be alanine(A) or threonine (T); amino acid #123 may be lysine (K), threonine (T)or alanine (A); amino acid #128 may be lysine (K) or threonine (T);amino acid #135 may be aspartic acid (D) or glutamic acid (E); aminoacid #136 may be alanine (A) or threonine (T); amino acid #145 may beglycine (G) or arginine (R); amino acid #149 may be glutamine (Q) orlysine (K); or any combination thereof.

PilA may consist of or comprise an amino acid sequence that differs fromSEQ ID NO. 58 at any one or more amino acid selected from the groupconsisting of amino acid #6, amino acid #7, amino acid #37, amino acid#44, amino acid #57, amino acid #67, amino acid #68, amino acid #69,amino acid #71, amino acid #73, amino acid #76, amino acid #84, aminoacid #86, amino acid #91, amino acid #94, amino acid #96, amino acid#97, amino acid #99, amino acid #103, amino acid #109, amino acid #110,amino acid #112, amino acid #114, amino acid #116, amino acid #118 aminoacid, #121, amino acid #122, amino acid #123, amino acid #128, aminoacid #135, amino acid #136, amino acid #145 and amino acid #149, whereinamino acid #6 is leucine (L); amino acid #7 is threonine (T); amino acid#37 is lysine (K); amino acid #44 is serine (S); amino acid #57 isserine (S); amino acid #67 is glycine (G); amino acid #68 is lysine (K);amino acid #69 is proline (P); amino acid #71 is lysine (K), serine (S)or threonine (T); amino acid #73 is serine (S) or methionine (M); aminoacid #76 is serine (S) or asparagine (N); amino acid #84 is lysine (K);amino acid #86 is valine (V); amino acid #91 is alanine (A); amino acid#94 is isoleucine (I) or lysine (K); amino acid #96 is glutamine (Q);amino acid #97 is serine (S); amino acid #99 is glycine (G); amino acid#103 is alanine (A); amino acid #109 is aspartic acid (D) or threonine(T); amino acid #110 is glycine (G) or arginine (R); amino acid #112 isserine (S); amino acid #114 is threonine (T); amino acid #116 isthreonine (T); amino acid #118 is glutamic acid (E), alanine (A), lysine(K) or serine (S); amino acid #121 is serine (S); amino acid #122 isthreonine (T); amino acid #123 is lysine (K) or alanine (A); amino acid#128 is lysine (K); amino acid #135 is glutamic acid (E); amino acid#136 is threonine (T); amino acid #145 is arginine (R); amino acid #149is lysine (K).

TABLE 2Pilin A amino acid sequences from 64 strains of Haemophilus influenzae (SEQID NO. 58 - SEQ ID NO. 121) Strain Name PilA sequence 86-028NPMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 58) NTHi3219CMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTKCTGGKNGIAADITTAKGYVKSVTTSNGAITVAGNGTLDGMSYTLTAEGDSAKGVTWKTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 59) NTHi3224AMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 60) NTHi12MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYKNYTKKAAVSELLQASAPYKADVELCVYSTGKPSSCSGGSNGIAADITTAKGYVASVITQSGGITVKGDGTLANMEYILQAAGNAAAGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 61) NTHi44MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 62) NTHi67MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKSDVELCVYSTGKPSTCSGGSNGIAADITTVKGYVKSVTTSNGAITVAGNGTLDGMSYTLTAEGDSAKGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 63) 1054MEEMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 64) 1729MEEMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 65) 1728MEEMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 66) 1885MEEMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYKNYTKKAAVSELLQASAPYKADVELCVYSTNEITNCMGGKNGIAADITTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAAAGVTWTTTCKGTDASLFPANFCGSITQ (SEQ ID NO. 67) 1060MEEMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKASVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQAKGNATAGVTWTTTCKGTDASLFPANFCRSVTK (SEQ ID NO. 68) RdKW20MKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTSCTGGKNGIAADIKTAKGYVASVITQSGGITVKGNGTLANMEYILQAKGNAAAGVTWTTTCKGTDASLFPANFCGSVTK (SEQ ID NO. 69) 214NPMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSSCSGGSNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQASGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 70) 1236MEEMKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTSCTGGKNGIAADIKTAKGYVASVITQSGGITVKGNGTLANMEYILQAKGNAAAGVTWTTTCKGTDASLFPANFCGSVTK (SEQ ID NO. 71) 1714MEEMKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 72) 1128MEEMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKASVSELLQASAPYKSDVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQAKGNATAGVTWTTTCKGTDASLFPANFCRSVTK (SEQ ID NO. 73) R2846MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 74) R2866MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTEASLFPANFCGSVTQ (SEQ ID NO. 75) 3655MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKASVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQAKGNATAGVTWTTTCKGTDASLFPANFCRSVTK (SEQ ID NO. 76) PittAAMKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 77) PittGGMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQAKGNATAGVTWTTTCKGTDASLFPANFCRSVTK (SEQ ID NO. 78) PittIIMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTEASLFPANFCGSVTQ (SEQ ID NO. 79) R3021MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTEASLFPANFCGSVTQ (SEQ ID NO. 80) 22.4-21MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKSDVELCVYSTGKPSTCSGGSNGIAADITTAKGYVKSVTTSNGAITVAGNGTLDGMSYTLTAEGDSAKGVTWKTTCKGTDASLFPANFCGSVTK (SEQ ID NO. 81) 3185AMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNEATKCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQASGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 82) 3221BMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNEATKCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQASGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 83) 3241AMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 84) 038144S1MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAISELLQASAPYKSDVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQAKGNATAGVTWTTTCKGTDASLFPANFCRSVTK (SEQ ID NO. 85) 821246MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTEASLFPANFCGSVTQ (SEQ ID NO. 86) 840645MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 87) 902550Z19MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKSDVELCVYSTGKPSTCSGGSNGIAADITTVKGYVKSVTTSNGAITVAGNGTLDGMSYTLTAEGDSAKGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 88) A840177MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 89) A920030MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 90) A950014MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGSNGIAADITTAKGYVKSVTTSNGAITVAGNGTLDRMSYTLTAEGDSAKGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 91) 901905UMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSSCSGGSNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQASGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 92) A920029MKLTTQTTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKSDVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVITQSGGITVKGNGTLTNMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSITQ (SEQ ID NO. 93) A930105MKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 94) 306543X4MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSSCSGGSNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQASGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 95) N218MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNEATKCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQASGNAATGVTWTTTCKGTDTSLFPANFCGSVTQ (SEQ ID NO. 96) N163MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 97) N162MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 98) N120MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQAKGNATAGVTWTTTCKGTDASLFPANFCRSVTK (SEQ ID NO. 99) N107MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQAKGNATAGVTWTTTCKGTDASLFPANFCRSVTK (SEQ ID NO. 100) N92MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 101) N91MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQAKGNATAGVTWTTTCKGTDASLFPANFCRSVTK (SEQ ID NO. 102) D219PGMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNEATKCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQASGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 103) D211PGMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 104) D211PDMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 105) D204CDMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILXATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 106) D198PGMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 107) D198PDMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 108) D195PDMKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 109) D195CDMKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 110) D189PGMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTSCTGGKNGIAADITTAKGYVKSVTTSNGAITVAGNGTLDGMSYTLTAEGDSAKGVTWKTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 111) D189PDMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTSCTGGKNGIAADITTAKGYVKSVTTSNGAITVAGNGTLDGMSYTLTAEGDSAKGVTWKTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 112) D124PGMKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 113) D124PDMKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 114) D124CGMKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 115) D58PGMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTEASLFPANFCGSVTQ (SEQ ID NO. 116) BS433MKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 117) BS432MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQAKGNATAGVTWTTTCKGTDASLFPANFCRSVTK (SEQ ID NO. 118) BS430MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNEATKCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQASGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 119) 1714MKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 120) 1128MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKASVSELLQASAPYKSDVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQAKGNATAGVTWTTTCKGTDASLFPANFCRSVTK (SEQ ID NO. 121)

PilA may be PilA from H. influenzae strain NTHi3219C, NTHi3224A, NTHi12,NTHi44, NTHi67, 1054MEE, 1729MEE, 1728MEE, 1885MEE, 1060MEE, RdKW20,214NP, 1236MEE, 1714MEE, 1128MEE, 86-028NP, R2846, R2866, 3655, PittAA,PittGG, PittII, R3021, 22.4-21, 3185A, 3221B, 3241A, 038144S1, 821246,840645, 902550Z19, A840177, A920030, A950014, 901905U, A920029, A930105,306543X4, N218, N163, N162, N120, N107, N92, N91, D219PG, D211PG,D211PD, D204CD, D198PG, D198PD, D195PD, D195CD, D189PG, D189PD, D124PG,D124PD, D124CG, D58PG, BS433, BS432, BS430, 1714 or 1128. An amino acidsequence for PilA from H. influenzae strain D204CD is set forth in SEQID NO. 106, wherein X at position #116 is either glutamine (Q) orleucine (L); ambiguity as to the amino acid at position #116 could becleared up by technical resolution of the second nucleotide encodingamino acid #116, clarifying the PilA sequence for strain D204CD. PilAmay be PilA as set forth in any of SEQ ID NO. 58-SEQ ID NO. 121.

PilA may be a sequence with at least 95%, 98%, or 99% identity, over theentire length, to any of SEQ ID NO. 58-SEQ ID NO. 121 (as set out inTable 2).

Immunogenic fragments of PilA comprise immunogenic fragments of at least7, 10, 15, 20, 25, 30 or 50 contiguous amino acids of SEQ ID NO. 58-SEQID NO. 121. The immunogenic fragments may elicit antibodies which canbind the full length sequence from which the fragment is derived. Theimmunogenic fragments may comprise a B and/or T cell epitope of SEQ IDNO. 58-SEQ ID NO:121.

For example, immunogenic fragments of PilA comprise immunogenicfragments of at least 7, 10, 15, 20, 25, 30 or 50 contiguous amino acidsof SEQ ID NO. 58. In one embodiment the immunogenic fragment of PilAcomprises less than 150, 125, 100, 75, or 60 amino acids of PilA, in afurther embodiment the immunogenic composition comprises less than 150,125, 100, 75 or 60 amino acids of PilA. The immunogenic fragments mayelicit antibodies which can bind SEQ ID NO. 58. The immunogenicfragments may comprise a B and/or T cell epitope of SEQ ID NO:58.

In one embodiment the immunogenic fragment of PilA is a fragment from H.influenzae strain 86-028NP wherein PilA is SEQ ID NO. 58.

PilA from H. influenzae strain 86-028NP - SEQ ID NO. 58MKLTTQQTLK KGFTLIELMI VIAIIAILAT IAIPSYQNYTKKAAVSELLQ ASAPYKADVE LCVYSTNETT NCTGGKNGIAADITTAKGYV KSVTTSNGAI TVKGDGTLAN MEYILQATGNAATGVTWTTT CKGTDASLFP ANFCGSVTQL

In another embodiment, the immunogenic fragment of PilA is approximatelyat least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to SEQ ID NO.127. More specifically, in one embodiment the immunogenic fragment ofPilA is SEQ ID NO. 127, a fragment consisting of amino acids 40-149 ofSEQ ID NO. 58.

Amino acids 40-149 of PilA from H. influenzaestrain 86-028NP - SEQ ID NO. 127.T KKAAVSELLQ ASAPYKADVE LCVYSTNETT NCTGGKNGIAADITTAKGYV KSVTTSNGAI TVKGDGTLAN MEYILQATGNAATGVTWTTT CKGTDASLFP ANFCGSVTQ

In another embodiment, the immunogenic fragment of PilA consists ofamino acids 40-149 from any of SEQ ID NO. 58-SEQ ID NO. 121. In anadditional embodiment, the immunogenic fragment is at least 95%identical to amino acids 40-149 from any of SEQ ID NO. 58-SEQ ID NO. 121

Identity between polypeptides may be calculated by various algorithms.For example, the Needle program, from the EMBOSS package (Free software;EMBOSS: The European Molecular Biology Open Software Suite (2000).Trends in Genetics 16(6): 276-277) and the Gap program from the GCG®package (Accelrys Inc.) may be used. This Gap program is animplementation of the Needleman-Wunsch algorithm described in:Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453. TheBLOSUM62 scoring matrix has been used, and the gap open and extensionpenalties were respectively 8 and 2.

Looking at the computed alignment, identical residues between twocompared sequences can be observed. A percentage of identity can becomputed by (1) calculating the number of identities divided by thelength of the alignment, multiplied by 100 (for example, for the Needleprogram analysis), (2) calculating the number of identities divided bythe length of the longest sequence, multiplied by 100, (3) calculatingthe number of identities divided by the length of the shortest sequence,multiplied by 100, or (4) calculating the number of identities dividedby the number of aligned residues, multiplied by 100 (a residue isaligned if it is in front of another) (for example, for the Gap programanalysis).

In one embodiment the PilA is capable of eliciting an immune responsewhich recognizes SEQ ID NO. 58.

Protein E/PilA Fusion Protein

In one embodiment Protein E and PilA are present in a fusion protein. Ina further embodiment the fusion protein has formula (I):L(X)_(m)—(R₁)_(n)-A-(Y)_(o)—B—(Z)_(p)  (formula I)wherein:X is a signal peptide or MHHHHHH (SEQ ID NO. 2);m is 0 or 1;R₁ is an amino acid;n is 0, 1, 2, 3, 4, 5 or 6;A is Protein E from Haemophilus influenzae or an immunogenic fragmentthereof, or PilA from Haemophilus influenzae or an immunogenic fragmentthereof;Y is selected from the group consisting of GG, SG, SS and (G)_(h)wherein h is 4, 5, 6, 7, 8, 9, or 10;o is 0 or 1;B is PilA from Haemophilus influenzae or an immunogenic fragmentthereof, or Protein E from Haemophilus influenzae or an immunogenicfragment thereof;Z is GGHHHHHH (SEQ ID NO: 3); andp is 0 or 1.

In one embodiment, the fusion proteins of formula (I) are definedwherein X is selected from the group consisting of the signal sequencefrom CcmH (cytochrome c membrane protein H), DsbA (periplasmic proteindisulfide isomerise I), DsbB (disulfide bond membrane protein B), FlgI(flagellar peptidoglycan ring protein), FocC (F1c Chaperone protein),MalE (maltose transporter subunit E), NadA (quinolinate synthase subunitA), NikA (nickel ABC transporter component A), NspA (Neisserial surfaceprotein A), Omp26 (outer membrane protein 26), OmpA (outer membraneprotein A), OspA (outer surface protein A), pelB (pectate lyase B), PhoA(bacterial alkaline phosphatase), PhtD (poly histidine triad protein D),PhtE (poly histidine triad protein E), SfmC (periplasmic pilinchaperone), Sip1 (surface immunogenic protein), TolB (Tol-Pal CellEnvelope Complex Component B), TorA (trimethylamine N-oxide reductasesystem subunit A), TorT (trimethylamine N-oxide reductase systemperiplasmic protein T) and YraI (putative periplasmic pilin chaperone);or any subgroup thereof. In one embodiment, X is a co-translationalsignal peptide or a post-translational signal peptide. In one embodimentX is the signal sequence from FlgI (flgI sp). In another particularembodiment, X is the signal sequence from pelB (pelB sp). In anotherembodiment, X is a post-translational signal peptide. In anotherembodiment, X is selected from the group consisting of the signalsequence from FlgI, NadA and pelB.

In one embodiment, the fusion proteins of formula (I) are definedwherein m is 1. In another embodiment, m is 0.

In one particular embodiment, R₁ and n are defined wherein (R₁)_(n) is 1to 6 amino acids enriched in small, usually hydrophilic, amino acids.Hydrophilic amino acids include glutamic acid (E), aspartic acid (D) andasparagine (N).

In one embodiment, the fusion proteins of formula (I) are definedwherein n is selected from the group consisting of 0, 1, 2 and 6. In oneparticular embodiment, R₁ and n are defined wherein (R₁)_(n) is selectedfrom the group consisting of D, E, ATNDDD (SEQ ID NO. 178) and MD, orany subset thereof.

In one particular embodiment, n is selected from the group consisting of1, 2 and 6. In one particular embodiment, n is 0.

In one embodiment, the fusion proteins of formula (I) are definedwherein A is Protein E from H. influenzae. In another embodiment, thefusion proteins of formula (I) are defined wherein A is Protein E asencoded by an amino acid sequence selected from the group consisting ofSEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8,SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO.13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ IDNO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27,SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO.32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ IDNO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41, SEQID NO. 42, SEQ ID NO. 43 SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46,SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO.51, SEQ ID NO. 52, SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, SEQ IDNO. 56 and SEQ ID NO. 57; or any subset of SEQ ID NO. 5 through SEQ IDNO. 57. In another embodiment, the fusion proteins of formula (I) aredefined wherein A is Protein E, wherein Protein E is approximately atleast 75%, 80%, 85%, 90%, 92%, 95%, 98% or 99% identical to the ProteinE amino acid sequence set forth in SEQ ID NO: 4. In another embodiment,A is Protein E wherein Protein E is approximately 90% to 100% identicalto the Protein E amino acid sequence set forth in SEQ ID NO: 4. Inanother embodiment, A is Protein E wherein Protein E is at least 95%identical to the Protein E amino acid sequence set forth in SEQ ID NO:4. In additional embodiment, A is Protein E wherein Protein E is atleast 95% identical to Protein E as set for in any of SEQ ID NO. 4-SEQID NO. 57. In a particular embodiment, A is Protein E having the aminoacid sequence set forth in SEQ ID NO. 4.

In another embodiment, the fusion proteins of formula (I) are definedwherein A is an immunogenic fragment of Protein E from H. influenzae. Inanother embodiment, A is an immunogenic fragment of Protein E whereinProtein E has an amino acid sequence selected from the group consistingof SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8,SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO.13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ IDNO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27,SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO.32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ IDNO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41, SEQID NO. 42, SEQ ID NO. 43 SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46,SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO.51, SEQ ID NO. 52, SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, SEQ IDNO. 56 and SEQ ID NO. 57; or any subset of SEQ ID NO. 4 through SEQ IDNO. 57. In another embodiment, A is an immunogenic fragment of ProteinE, wherein Protein E is approximately 75%, 80%, 85%, 90%, 92%, 95%, 98%or 99% identical to the amino acid sequence set forth in SEQ ID NO: 4.In another embodiment, A is an immunogenic fragment of Protein E,wherein Protein E is approximately 90% to 100% identical to SEQ ID NO.4. In an additional embodiment, A is an immunogenic fragment of ProteinE, wherein Protein E is at least 95% identical to any of SEQ ID NO.4-SEQ ID NO. 57. More specifically, in one embodiment, A is animmunogenic fragment of Protein E, wherein Protein E is at least 93%,95%, 98%, 99% or 100% identical to SEQ ID NO. 124. In a particularembodiment, A is an immunogenic fragment of Protein E wherein Protein Eis SEQ ID NO. 4.

In another embodiment, A is an immunogenic fragment of Protein E from H.influenzae selected from the group consisting of amino acids 17-160 ofSEQ ID NO. 4 (SEQ ID NO. 122), amino acids 18-160 of SEQ ID NO. 4 (SEQID NO. 123), amino acids 19-160 of SEQ ID NO. 4 (SEQ ID NO. 124), aminoacids 20-160 of SEQ ID NO. 4 (SEQ ID NO. 125) and amino acids 22-160 ofSEQ ID NO. 4 (SEQ ID NO. 126). In another embodiment, A is animmunogenic fragment of Protein E from H. influenzae selected from thegroup consisting of amino acids 17-160 of SEQ ID NO. 4 (SEQ ID NO. 122),amino acids 18-160 of SEQ ID NO. 4 (SEQ ID NO. 123), amino acids 19-160of SEQ ID NO. 4 (SEQ ID NO. 124), amino acids 20-160 of SEQ ID NO. 4(SEQ ID NO. 125), amino acids 22-160 of SEQ ID NO. 4 (SEQ ID NO. 126),amino acids 23-160 of SEQ ID NO. 4 (SEQ ID NO. 179) and amino acids24-160 of SEQ ID NO. 4 (SEQ ID NO. 180). In a further embodiment, A isan immunogenic fragment of Protein E from H. influenzae selected fromthe group consisting of amino acids 17-160 of SEQ ID NO. 4 (SEQ ID NO.122), amino acids 18-160 of SEQ ID NO. 4 (SEQ ID NO. 123), amino acids20-160 of SEQ ID NO. 4 (SEQ ID NO. 125), amino acids 22-160 of SEQ IDNO. 4 (SEQ ID NO. 126), amino acids 23-160 of SEQ ID NO. 4 (SEQ ID NO.179) and amino acids 24-160 of SEQ ID NO. 4 (SEQ ID NO. 180). Morespecifically, in one embodiment, A is SEQ ID NO. 124, amino acids 19-160of SEQ ID NO. 4. In an additional embodiment, A is SEQ ID NO. 125, aminoacids 20-160 of SEQ ID NO. 5. In another embodiment, A is immunogenicfragment of Protein E from H. influenzae selected from the groupconsisting of amino acids 23-160 of SEQ ID NO. 4 (SEQ ID NO. 179) andamino acids 24-160 of SEQ ID NO. 4 (SEQ ID NO. 180).

Protein E - SEQ ID NO. 4 MKKIILTLSL GLLTACSAQI QKAEQNDVKL APPTDVRSGYIRLVKNVNYY IDSESIWVDN QEPQIVHFDA VVNLDKGLYVYPEPKRYARS VRQYKILNCA NYHLTQVRTD FYDEFWGQGLRAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKKAmino acids 17-160 of Protein E from SEQ ID NO.  4 - SEQ ID NO. 122SAQI QKAEQNDVKL APPTDVRSGY IRLVKNVNYY IDSESIWVDNQEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCANYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKKAmino acids 18-160 of Protein E from SEQ ID NO. 4 - SEQ ID NO. 123AQI QKAEQNDVKL APPTDVRSGY IRLVKNVNYY IDSESIWVDNQEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCANYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKKAmino acids 19-160 of Protein E from SEQ ID NO. 4 - SEQ ID NO. 124QI QKAEQNDVKL APPTDVRSGY IRLVKNVNYY IDSESIWVDNQEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCANYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKKAmino acids 20-160 of Protein E from SEQ ID NO. 4 - SEQ ID NO. 125I QKAEQNDVKL APPTDVRSGY IRLVKNVNYY IDSESIWVDNQEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCANYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKKAmino acids 22-160 of Protein E from SEQ ID NO. 4 - SEQ ID NO. 126KAEQNDVKL APPTDVRSGY IRLVKNVNYY IDSESIWVDNQEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCANYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKKAmino acids 23-160 of Protein E from SEQ ID NO. 4 - SEQ ID NO. 179AEQNDVKL APPTDVRSGY IRLVKNVNYY IDSESIWVDNQEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCANYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKKAmino acids 24-160 Protein E from SEQ ID NO. 4 - SEQ ID NO. 180EQNDVKL APPTDVRSGY IRLVKNVNYY IDSESIWVDNQEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCANYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKK

In another embodiment, the fusion proteins of formula (I) are definedwherein A is PilA from H. influenzae. In another embodiment, the fusionproteins of formula (I) are defined wherein A is PilA from H. influenzaehaving an amino acid sequence selected from the group consisting of SEQID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62,SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO.67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71, SEQ IDNO. 72, SEQ ID NO. 73, SEQ ID NO. 74, SEQ ID NO. 75, SEQ ID NO. 76, SEQID NO. 77, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO. 80, SEQ ID NO. 81,SEQ ID NO. 82, SEQ ID NO. 83, SEQ ID NO. 84, SEQ ID NO. 85, SEQ ID NO.86, SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 89, SEQ ID NO. 90, SEQ IDNO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO. 94, SEQ ID NO. 95, SEQID NO. 96, SEQ ID NO. 97, SEQ ID NO. 98, SEQ ID NO. 99, SEQ ID NO. 100,SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103, SEQ ID NO. 104, SEQ IDNO. 105, SEQ ID NO. 106, SEQ ID NO. 107, SEQ ID NO. 108, SEQ ID NO. 109,SEQ ID NO. 110, SEQ ID NO. 111, SEQ ID NO. 112, SEQ ID NO. 113, SEQ IDNO. 114, SEQ ID NO. 115, SEQ ID NO. 116, SEQ ID NO. 117, SEQ ID NO. 118,SEQ ID NO. 119, SEQ ID NO. 120 and SEQ ID NO. 121; or any subset of SEQID NO. 58 through SEQ ID NO. 121. In another embodiment, A is PilAwherein PilA is approximately at least 75%, 80%, 85%, 90%, 92%, 95%, 98%or 99% identical to SEQ ID NO. 58. In another embodiment, A is PilAwherein PilA is at least 95% identical to any of SEQ ID NO. 58-SEQ IDNO. 121. In a particular embodiment, A is PilA of SEQ ID NO. 58.

In another embodiment, the fusion proteins of formula (I) are definedwherein A is an immunogenic fragment of PilA from H. influenzae. Inanother embodiment, A is an immunogenic fragment of PilA wherein PilA isapproximately at least 75%, 80%, 85%, 90%, 92%, 95%, 98% or 99%identical to SEQ ID NO. 58. For example, A is an immunogenic fragment ofPilA wherein PilA has an amino acid sequence selected from the groupconsisting of SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO.61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ IDNO. 66, SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQID NO. 71, SEQ ID NO. 72, SEQ ID NO. 73, SEQ ID NO. 74, SEQ ID NO. 75,SEQ ID NO. 76, SEQ ID NO. 77, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO.80, SEQ ID NO. 81, SEQ ID NO. 82, SEQ ID NO. 83, SEQ ID NO. 84, SEQ IDNO. 85, SEQ ID NO. 86, SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 89, SEQID NO. 90, SEQ ID NO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO. 94,SEQ ID NO. 95, SEQ ID NO. 96, SEQ ID NO. 97, SEQ ID NO. 98, SEQ ID NO.99, SEQ ID NO. 100, SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103, SEQID NO. 104, SEQ ID NO. 105, SEQ ID NO. 106, SEQ ID NO. 107, SEQ ID NO.108, SEQ ID NO. 109, SEQ ID NO. 110, SEQ ID NO. 111, SEQ ID NO. 112, SEQID NO. 113, SEQ ID NO. 114, SEQ ID NO. 115, SEQ ID NO. 116, SEQ ID NO.117, SEQ ID NO. 118, SEQ ID NO. 119, SEQ ID NO. 120 and SEQ ID NO. 121;or any subset SEQ ID NO. 58 through SEQ ID NO. 121. In an additionalembodiment, A is an immunogenic fragment of PilA wherein PilA is atleast 95% identical to any of SEQ ID NO. 58-SEQ ID NO. 121. In aparticular embodiment, A is an immunogenic fragment of PilA from H.influenzae strain 86-028NP wherein PilA is SEQ ID NO. 58.

PilA from H. influenzae strain 86-028NP - SEQ ID NO. 58MKLTTQQTLK KGFTLIELMI VIAIIAILAT IAIPSYQNYTKKAAVSELLQ ASAPYKADVE LCVYSTNETT NCTGGKNGIAADITTAKGYV KSVTTSNGAI TVKGDGTLAN MEYILQATGNAATGVTWTTT CKGTDASLFP ANFCGSVTQL

In another embodiment, A is an immunogenic fragment of PilAapproximately at least 75%, 80%, 85%, 90%, 92%, 95%, 98% or 99%identical to SEQ ID NO. 127. More specifically, in one embodiment A isSEQ ID NO. 127, a fragment consisting of amino acids 40-149 of SEQ IDNO. 58.

Amino acids 40-149 of PilA from H. influenzaestrain 86-028NP - SEQ ID NO. 127.T KKAAVSELLQ ASAPYKADVE LCVYSTNETT NCTGGKNGIAADITTAKGYV KSVTTSNGAI TVKGDGTLAN MEYILQATGNAATGVTWTTT CKGTDASLFP ANFCGSVTQ

In another embodiment, A is an immunogenic fragment of PilA consistingof amino acids 40-149 from any of SEQ ID NO. 58-SEQ ID NO. 121. In anadditional embodiment, A is an immunogenic fragment at least 95%identical to amino acids 40-149 from any of SEQ ID NO. 58-SEQ ID NO.121.

In one embodiment, the fusion proteins of formula (I) are definedwherein Y is selected from the group consisting of GG, SG and SS. Inanother embodiment, the fusion proteins of formula (I) are definedwherein Y is GG or SG. In one particular embodiment, Y is GG.

In one embodiment, the fusion proteins of formula (I) are definedwherein o is 1. In another embodiment, o is 0.

In one embodiment, the fusion proteins of formula (I) are definedwherein B is PilA from H. influenzae or an immunogenic fragment of PilAfrom H. influenzae when A is Protein E from H. influenzae or animmunogenic fragment of Protein E from H. influenzae. For example, B isPilA from H. influenzae strain 86-028NP. In another embodiment, B isPilA from H. influenzae having an amino acid sequence selected from thegroup consisting of SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ IDNO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70,SEQ ID NO. 71, SEQ ID NO. 72, SEQ ID NO. 73, SEQ ID NO. 74, SEQ ID NO.75, SEQ ID NO. 76, SEQ ID NO. 77, SEQ ID NO. 78, SEQ ID NO. 79, SEQ IDNO. 80, SEQ ID NO. 81, SEQ ID NO. 82, SEQ ID NO. 83, SEQ ID NO. 84, SEQID NO. 85, SEQ ID NO. 86, SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 89,SEQ ID NO. 90, SEQ ID NO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO.94, SEQ ID NO. 95, SEQ ID NO. 96, SEQ ID NO. 97, SEQ ID NO. 98, SEQ IDNO. 99, SEQ ID NO. 100, SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103,SEQ ID NO. 104, SEQ ID NO. 105, SEQ ID NO. 106, SEQ ID NO. 107, SEQ IDNO. 108, SEQ ID NO. 109, SEQ ID NO. 110, SEQ ID NO. 111, SEQ ID NO. 112,SEQ ID NO. 113, SEQ ID NO. 114, SEQ ID NO. 115, SEQ ID NO. 116, SEQ IDNO. 117, SEQ ID NO. 118, SEQ ID NO. 119, SEQ ID NO. 120 and SEQ ID NO.121; or any subset of SEQ ID NO. 58 through SEQ ID NO. 121. In anotherembodiment, B is PilA wherein PilA is approximately at least 75%, 80%,85%, 90%, 92%, 95%, 98% or 99% identical to SEQ ID NO. 58. In anotherembodiment, B is PilA wherein PilA is at least 95%, 98% or 99% identicalto any of SEQ ID NO. 58-SEQ ID NO. 121. In a particular embodiment, B isPilA of SEQ ID NO. 58.

In another embodiment, B is PilA wherein PilA is at least 95%, 98% or99% identical to any of SEQ ID NO. 58-SEQ ID NO. 121 and A is PE whereinPE is at least 95%, 98% or 99% identical to any of SEQ ID NO. 4-SEQ IDNO. 57.

In another embodiment, the fusion proteins of formula (I) are definedwherein B is an immunogenic fragment of PilA from H. influenzae when Ais an immunogenic fragment of Protein E from H. influenzae. For example,B is an immunogenic fragment of the PilA from H. influenzae strain86-028NP. In another embodiment, B is an immunogenic fragment of PilAwherein PilA is approximately at least 80%, 85%, 90%, 95%, 98% or 99%identical to SEQ ID NO: 58. In another embodiment, B is an immunogenicfragment of PilA wherein PilA has an amino acid selected from the groupconsisting of SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO.61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ IDNO. 66, SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQID NO. 71, SEQ ID NO. 72, SEQ ID NO. 73, SEQ ID NO. 74, SEQ ID NO. 75,SEQ ID NO. 76, SEQ ID NO. 77, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO.80, SEQ ID NO. 81, SEQ ID NO. 82, SEQ ID NO. 83, SEQ ID NO. 84, SEQ IDNO. 85, SEQ ID NO. 86, SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 89, SEQID NO. 90, SEQ ID NO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO. 94,SEQ ID NO. 95, SEQ ID NO. 96, SEQ ID NO. 97, SEQ ID NO. 98, SEQ ID NO.99, SEQ ID NO. 100, SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103, SEQID NO. 104, SEQ ID NO. 105, SEQ ID NO. 106, SEQ ID NO. 107, SEQ ID NO.108, SEQ ID NO. 109, SEQ ID NO. 110, SEQ ID NO. 111, SEQ ID NO. 112, SEQID NO. 113, SEQ ID NO. 114, SEQ ID NO. 115, SEQ ID NO. 116, SEQ ID NO.117, SEQ ID NO. 118, SEQ ID NO. 119, SEQ ID NO. 120 and SEQ ID NO. 121;or any subset of SEQ ID NO. 58 through SEQ ID NO. 121. In anotherembodiment, B is an immunogenic fragment of PilA wherein PilA is atleast 95%, 98% or 99% identical to any of SEQ ID NO. 58-SEQ ID NO. 121.In a particular embodiment, B is an immunogenic fragment of PilA from H.influenzae wherein PilA has the amino acid sequence set forth in SEQ IDNO. 58. In another embodiment, B is an immunogenic fragment of PilAconsisting of amino acids 40-149 from any of SEQ ID NO. 58-SEQ ID NO.121. More specifically, in one embodiment B is the fragment of PilA asset forth in SEQ ID NO. 127. In an additional embodiment, B is animmunogenic fragment at least 95%, 98% or 99% identical to amino acids40-149 of any of SEQ ID NO. 58-SEQ ID NO. 121.

In one particular embodiment, B is the fragment of PilA as set forth inSEQ ID NO. 127 and A is an immunogenic fragment of Protein E selectedfrom the group consisting of SEQ ID NO. 122, SEQ ID NO. 124, SEQ ID NO.125 and SEQ ID NO. 126. More particularly, B is the fragment of PilA asset forth in SEQ ID NO. 127 and A is the fragment of Protein E as setforth in SEQ ID NO. 124, amino acids 19-160 of Protein E from SEQ ID NO.4. In another embodiment, B is the fragment of PilA as set forth in SEQID NO. 127 and A is the fragment of Protein E as set forth in SEQ ID NO.125.

In another embodiment, B is an immunogenic fragment of PilA wherein PilAis at least 95% identical to any of SEQ ID NO. 58-SEQ ID NO. 121 and Ais an immunogenic fragment of PE wherein PE is at least 95% identical toany of SEQ ID NO. 4-SEQ ID NO. 57.

In another embodiment, the fusion proteins of formula (I) are definedwherein B is Protein E from H. influenzae when A is PilA from H.influenzae. For example, B is Protein E having an amino acid sequenceselected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 5, SEQ IDNO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ IDNO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20,SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO.25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ IDNO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39,SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43 SEQ ID NO.44, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48, SEQ IDNO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 53, SEQID NO. 54, SEQ ID NO. 55, SEQ ID NO. 56 and SEQ ID NO. 57; or any subsetof SEQ ID NO. 4 through SEQ ID NO. 57. In another embodiment, the fusionproteins of formula (I) are defined wherein B is Protein E whereinProtein E is approximately at least 75%, 80%, 85%, 90%, 95%, 98% or 99%identical to the Protein E amino acid sequence set forth in SEQ ID NO:4. In another embodiment, B is Protein E wherein Protein E isapproximately 90%, 95%, 98%, or 99% identical to the Protein E aminoacid sequence set forth in SEQ ID NO: 4. For example, B is Protein Ewherein Protein E is at least 95% identical to Protein E as set forth inSEQ ID NO. 4. In another embodiment, B is Protein E wherein Protein E isat least 95% identical to any of SEQ ID NO. 4-SEQ ID NO. 57. In aparticular embodiment, B is Protein E having the amino acid sequence setforth in SEQ ID NO. 4.

In another embodiment, the fusion proteins of formula (I) are definedwherein B is an immunogenic fragment of Protein E from H. influenzaewhen A is an immunogenic fragment of PilA from H. influenzae. Forexample, B is an immunogenic fragment of Protein E wherein Protein E hasan amino acid sequence selected from the group consisting of SEQ ID NO.4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9,SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO.14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ IDNO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28,SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO.33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ IDNO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQID NO. 43, SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47,SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO.52, SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO. 56 and SEQID NO. 57; or any subset of SEQ ID NO. 4 through SEQ ID NO. 57. Inanother embodiment, the fusion proteins of formula (I) are definedwherein B is an immunogenic fragment of Protein E wherein Protein E isapproximately at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical tothe Protein E amino acid sequence set forth in SEQ ID NO. 4. In anotherembodiment, B is an immunogenic fragment of Protein E wherein Protein Eis approximately 90% to 100% identical to the Protein E amino acidsequence set forth in SEQ ID NO: 4. In a particular embodiment, B is animmunogenic fragment of Protein E having the amino acid sequence setforth in SEQ ID NO. 4. In an additional embodiment, B is an immunogenicfragment of Protein E, wherein Protein E is at least 95% identical toany of SEQ ID NO. 4-SEQ ID NO. 57.

In another embodiment, B is a fragment of Protein E from H. influenzaeselected from the group consisting of amino acids 17-160 of SEQ ID NO. 4(SEQ ID NO. 122), amino acids 18-160 of SEQ ID NO. 4 (SEQ ID NO. 123),amino acids 19-160 of SEQ ID NO. 4 (SEQ ID NO. 124), amino acids 20-160of SEQ ID NO. 4 (SEQ ID NO. 125) and amino acids 22-160 of SEQ ID NO. 4(SEQ ID NO. 126). In another embodiment, B is an immunogenic fragment ofProtein E from H. influenzae selected from the group consisting of aminoacids 17-160 of SEQ ID NO. 4 (SEQ ID NO. 122), amino acids 18-160 of SEQID NO. 4 (SEQ ID NO. 123), amino acids 19-160 of SEQ ID NO. 4 (SEQ IDNO. 124), amino acids 20-160 of SEQ ID NO. 4 (SEQ ID NO. 125), aminoacids 22-160 of SEQ ID NO. 4 (SEQ ID NO. 126), amino acids 23-160 of SEQID NO. 4 (SEQ ID NO. 179) and amino acids 24-160 of SEQ ID NO. 4 (SEQ IDNO. 180). More specifically, in one embodiment, B is the fragment ofProtein E as set forth in SEQ ID NO. 123, amino acids 18-160 of SEQ IDNO. 4.

In one particular embodiment B is an immunogenic fragment of Protein Eas set forth in SEQ ID NO. 123, amino acids 18-160 of SEQ ID NO. 4, whenA is an immunogenic fragment of PilA as set forth in SEQ ID NO. 127.

In one embodiment, the fusion proteins of formula (I) are definedwherein p is 0. In another embodiment, the fusion proteins of formula(I) are defined wherein p is 1.

In one embodiment, the fusion protein of formula (I) is selected fromthe group consisting of SEQ ID NO. 136, SEQ ID NO. 138, SEQ ID NO. 140,SEQ ID NO. 142, SEQ ID NO. 144, SEQ ID NO. 146, SEQ ID NO. 148, SEQ IDNO. 150, SEQ ID NO. 182, SEQ ID NO. 184, SEQ ID NO. 186, SEQ ID NO. 188,SEQ ID NO. 190, SEQ ID NO. 192, SEQ ID NO. 194, SEQ ID NO. 196, SEQ IDNO. 198, SEQ ID NO. 200, SEQ ID NO. 202 and SEQ ID NO. 204; or anysubset thereof. In another embodiment, the fusion protein of formula (I)is approximately 85%, 88%, 90%, 92%, 95% or 98% identical to any of SEQID NO. 136, SEQ ID NO. 138, SEQ ID NO. 140, SEQ ID NO. 142, SEQ ID NO.144, SEQ ID NO. 146, SEQ ID NO. 148, SEQ ID NO. 150, SEQ ID NO. 182, SEQID NO. 184, SEQ ID NO. 186, SEQ ID NO. 188, SEQ ID NO. 190, SEQ ID NO.192, SEQ ID NO. 194, SEQ ID NO. 196, SEQ ID NO. 198, SEQ ID NO. 200, SEQID NO. 202 or SEQ ID NO. 204.

In one embodiment the fusion protein formula (I) is the fusion proteinof SEQ ID NO. 148 wherein the signal peptide has been removed SEQ ID NO.177 (QIQKAEQN DVKLAPPTDV RSGYIRLVKN VNYYIDSESI WVDNQEPQIV HFDAWNLDKGLYVYPEPKR YARSVRQYKI LNCANYHLTQ VRTDFYDEFW GQGLRAAPKK QKKHTLSLTPDTTLYNAAQI ICANYGEAFS VDKKGGTKKA AVSELLQASA PYKADVELCV YSTNETTNCTGGKNGIAADI TTAKGYVKSV TTSNGAITVK GDGTLANMEY ILQATGNAAT GVTWTTTCKGTDASLFPANF CGSVTQ).

In one embodiment the fusion protein of formula (I) is the fusionprotein of SEQ ID NO. 194 wherein the signal peptide has been removedSEQ ID NO. 219 (IQKAEQND VKLAPPTDVR SGYIRLVKNV NYYIDSESIW VDNQEPQIVHFDAWNLDKG LYVYPEPKRY ARSVRQYKIL NCANYHLTQV RTDFYDEFWG QGLRAAPKKQKKHTLSLTPD TTLYNAAQII CANYGEAFSV DKKGGTKKAA VSELLQASAP YKADVELCVYSTNETTNCTG GKNGIAADIT TAKGYVKSVT TSNGAITVKG DGTLANMEYI LQATGNAATGVTWTTTCKGT DASLFPANFC GSVTQ).

Streptococcus pneumoniae Capsular Saccharide Conjugates

The term capsular saccharide includes capsular polysaccharides andoligosaccharides derivable from the capsular polysaccharide. Anoligosaccharide contains at least 4 sugar residues. The terms conjugateand conjugated relate to a capsular saccharide covalently bonded to acarrier protein.

In an immunogenic composition, the total number of saccharide serotypesis optionally less than or equal to 23. In one embodiment theimmunogenic composition comprises less than 23, 22, 21, 20, 19, 18, 17,16, 15, 14, or 13 Streptococcus pneumoniae saccharides, optionally theimmunogenic composition comprising 10-23 serotypes, 10-16 serotypes,10-15 serotypes, 10-14 serotypes, 10-13 serotypes or 10-12 serotypes.

In one embodiment the Streptococcus pneumoniae capsular saccharideconjugates will be derived from the following serotypes 1, 2, 3, 4, 5,6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20,22F, 23F and 33F, although it is appreciated that one or two otherserotypes could be substituted depending on the age of the recipientreceiving the vaccine and the geographical location where theimmunogenic composition will be administered. For example, a 7 valentimmunogenic composition may comprise saccharides from serotypes 4, 6B,9V, 14, 18C, 19F and 23F. A 10 valent immunogenic composition mayfurther comprise saccharides derived from serotypes 1, 5 and 7F. A 12valent immunogenic composition may further comprise saccharides derivedfrom serotypes 6A, 19A. A 15 valent immunogenic composition may furthercomprise saccharides derived from serotypes 22F and 33F.

Further saccharide antigens, for example 23 valent (such as serotypes 1,2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A,19F, 20, 22F, 23F and 33F), are also contemplated by the invention.

The term “carrier protein” is intended to cover both small peptides andlarge polypeptides (>10 kDa). The carrier protein may be any peptide orprotein. It may comprise one or more T-helper epitopes. The carrierprotein may be tetanus toxoid (TT), tetanus toxoid fragment C, non-toxicmutants of tetanus toxin [note all such variants of TT are considered tobe the same type of carrier protein for the purposes of this invention],polypeptides comprising tetanus toxin T-cell epitopes such as N19(WO2006/067632), diphtheria toxoid (DT), CRM197 (cross reacting material197), other non-toxic mutants of diphtheria toxin [such as CRM176, CRM197, CRM228, CRM 45 (Uchida et al J. Biol. Chem. 218; 3838-3844, 1973);CRM 9, CRM 45, CRM102, CRM 103 and CRM107 (where CRM stands for crossreacting material) and other mutations described by Nicholls and Youlein Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992;deletion or mutation of Glu-148 to Asp, Gln or Ser and/or Ala 158 to Glyand other mutations disclosed in U.S. Pat. No. 4,709,017 or U.S. Pat.No. 4,950,740; mutation of at least one or more residues Lys 516, Lys526, Phe 530 and/or Lys 534 and other mutations disclosed in U.S. Pat.No. 5,917,017 or U.S. Pat. No. 6,455,673; or fragment disclosed in U.S.Pat. No. 5,843,711] (note all such variants of DT are considered to bethe same type of carrier protein for the purposes of this invention),pneumococcal pneumolysin (Kuo et al (1995) Infect Immun 63; 2706-13),OMPC (outer membrane protein C from meningococcus usually extracted fromN. meningitidis serogroup B-EP0372501), synthetic peptides (EP0378881,EP0427347), heat shock proteins (WO 93/17712, WO 94/03208), pertussisproteins (WO 98/58668, EP0471177), cytokines, lymphokines, growthfactors or hormones (WO 91/01146), artificial proteins comprisingmultiple human CD4+ T cell epitopes from various pathogen derivedantigens (Falugi et al (2001) Eur J Immunol 31; 3816-3824) such as N19protein (Baraldoi et al (2004) Infect Immun 72; 4884-7), pneumococcalsurface protein PspA (WO 02/091998), iron uptake proteins (WO 01/72337),toxin A or toxin B of Clostridium difficile (WO 00/61761), H. influenzaeProtein D (EP594610 and WO 00/56360), pneumococcal PhtA (WO 98/18930,also referred to Sp36), pneumococcal PhtD (poly histidine triad Ddisclosed in WO 00/37105, and is also referred to Sp036D), pneumococcalPhtB (poly histidine triad B disclosed in WO 00/37105, and is alsoreferred to Sp036B), or PhtE (poly histidine triad E disclosed inWO00/30299 and is referred to as BVH-3).

In one embodiment the Streptococcus pneumoniae capsular saccharideconjugates are conjugated to a carrier protein independently selectedfrom the group consisting of tetanus toxoid (TT), fragment C of TT,diphtheria toxoid, CRM197 (cross reacting material 197), detoxifiedpneumolysin, protein D (from H. influenzae), PhtD, PhtDE (a proteincontaining poly histidine triad protein D and poly histidine triadprotein E) and N19. In a further embodiment the Streptococcus pneumoniaecapsular saccharide conjugates are all independently conjugated toCRM197.

The term ‘conjugated to’ in this context means that the protein iscovalently bonded to a saccharide; in this situation the protein isacting as a carrier protein.

In one embodiment the immunogenic composition comprises at least oneStreptococcus pneumoniae capsular saccharide conjugated to protein D. Inone embodiment a minority of conjugated Streptococcus pneumoniaesaccharides are conjugated to protein D wherein the term ‘minority’refers to less than half of the total number of saccharides in thecomposition being conjugated to protein D. In a further embodiment theimmunogenic composition comprises between 1-20, 1-18, 1-16, 1-14, 1-12,1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-4 or 1-2 Streptococcus pneumoniaecapsular saccharide conjugated to protein D. In one embodiment theimmunogenic composition comprises at least one Streptococcus pneumoniaecapsular saccharides conjugated to diphtheria toxoid. In a furtherembodiment the immunogenic composition comprises 19F is conjugated todiphtheria toxoid. In one embodiment the immunogenic compositioncomprises at least one Streptococcus pneumoniae capsular saccharidesconjugated to tetanus toxoid. In a further embodiment the immunogeniccomposition comprises 18C is conjugated to tetanus toxoid.

In one embodiment the immunogenic composition comprises a conjugatedserotype 1 saccharide conjugated to protein D or CRM197. In oneembodiment the immunogenic composition comprises a conjugated serotype 4saccharide conjugated to protein D or CRM197. In one embodiment theimmunogenic composition comprises a conjugated serotype 5 saccharidewherein the serotype 5 saccharide is conjugated to protein D or CRM197.In one embodiment the immunogenic composition comprises a conjugatedserotype 6B saccharide wherein the serotype 6B saccharide is conjugatedto protein D or CRM197. In one embodiment the immunogenic compositioncomprises a conjugated serotype 7F saccharide is wherein the serotype 7Fsaccharide is conjugated to protein D or CRM197. In one embodiment theimmunogenic composition comprises a conjugated serotype 9V saccharidewherein the 9V saccharide is conjugated to protein D or CRM197. In oneembodiment the immunogenic composition comprises a conjugated serotype14 saccharide wherein the serotype 14 saccharide is conjugated toprotein D or CRM197. In one embodiment the immunogenic compositioncomprises a conjugated serotype 18C saccharide wherein the serotype 18Csaccharide is conjugated to tetanus toxoid or CRM197. In one embodimentthe immunogenic composition comprises a conjugated 19F saccharidewherein the serotype 19F saccharide is conjugated to diphtheria toxoidor CRM197. In one embodiment the immunogenic composition comprises aconjugated 23F saccharide wherein the serotype 23F saccharide isconjugated to protein D or CRM197. In one embodiment the immunogeniccomposition comprises a conjugated 6A saccharide conjugated to CRM197.In one embodiment the immunogenic composition comprises a conjugated 19Asaccharide conjugated to CRM197.

In one embodiment the immunogenic composition comprises a Streptococcuspneumoniae serotype 1 saccharide conjugated to protein D, aStreptococcus pneumoniae serotype 4 saccharide conjugated to protein D,a Streptococcus pneumoniae serotype 5 saccharide conjugated to proteinD, a Streptococcus pneumoniae serotype 6B saccharide conjugated toprotein D, a Streptococcus pneumoniae serotype 7F saccharide conjugatedto protein D, a Streptococcus pneumoniae serotype 9V saccharideconjugated to protein D, a Streptococcus pneumoniae serotype 14saccharide conjugated to protein D, a Streptococcus pneumoniae serotype23F saccharide conjugated to protein D, a Streptococcus pneumoniaeserotype 18C saccharide conjugated to tetanus toxoid and a Streptococcuspneumoniae 19F saccharide conjugated to diphtheria toxoid. In oneembodiment the immunogenic composition further comprises a Streptococcuspneumoniae serotype 6A conjugated to CRM197 and a Streptococcuspneumoniae serotype 19A conjugated to CRM197.

Optionally the ratio of carrier protein to S. pneumoniae saccharide isbetween 1:5 and 5:1; 1:2 and 2.5:1; 1:1 and 2:1 (w/w). In an embodiment,the majority of the conjugates, for example 6, 7, 8, 9 or more of theconjugates have a ratio of carrier protein to saccharide that is greaterthan 1:1, for example 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1 or 1.6:1.

In general, the immunogenic composition of the invention may comprise adose of each saccharide conjugate between 0.1 and 20 μg, 1 and 5 μg, 1and 10 μg or 1 and 3 μg of saccharide.

In an embodiment, the immunogenic composition of the invention containseach S. pneumoniae capsular saccharide conjugate at a dose of between0.1-20 μg; 0.5-10 μg; 0.5-5 μg or 1-3 μg of saccharide. In anembodiment, capsular saccharides may be present at different dosages,for example some capsular saccharides may be present at a dose ofexactly 1 μg or some capsular saccharides may be present at a dose ofexactly 3 μg. In an embodiment, saccharides from serotypes 3, 18C and19F (or 4, 18C and 19F) are present at a higher dose than othersaccharides. In one aspect of this embodiment, serotypes 3, 18C and 19F(or 4, 18C and 19F) are present at a dose of around or exactly 3 μgwhilst other saccharides in the immunogenic composition are present at adose of around or exactly 1 μg. In one embodiment serotypes 1, 5, 6B,7F, 9V, 14 and 23F are present at a dose of around or exactly 1 μg.

The term “saccharide” throughout this specification may indicatepolysaccharide or oligosaccharide and includes both. Polysaccharides areisolated from bacteria and may be sized to some degree by known methods(see for example EP497524 and EP497525) and optionally bymicrofluidisation. Polysaccharides can be sized in order to reduceviscosity in polysaccharide samples and/or to improve filterability forconjugated products. Oligosaccharides have a low number of repeat units(typically 5-30 repeat units) and are typically hydrolysedpolysaccharides.

Capsular polysaccharides of Streptococcus pneumoniae comprise repeatingoligosaccharide units which may contain up to 8 sugar residues. For areview of the oligosaccharide units for the key Streptococcus pneumoniaeserotypes see JONES, Christopher. Vaccines based on the cell surfacecarbohydrates of pathogenic bacteria. An. Acad. Bras. Ciênc., June 2005,vol. 77, no. 2, p. 293-324. ISSN 0001-3765. In one embodiment, acapsular saccharide antigen may be a full length polysaccharide, howeverin others it may be one oligosaccharide unit, or a shorter than nativelength saccharide chain of repeating oligosaccharide units. In oneembodiment, all of the saccharides present in the vaccine arepolysaccharides. Full length polysaccharides may be “sized” i.e. theirsize may be reduced by various methods such as acid hydrolysistreatment, hydrogen peroxide treatment, sizing by Emulsiflex® followedby a hydrogen peroxide treatment to generate oligosaccharide fragmentsor microfluidization.

In one embodiment the immunogenic composition further comprisesunconjugated S. penumoniae saccharides of serotypes different from thoseconjugated, such that the number of conjugated and unconjugatedsaccharide serotypes is less than or equal to 23.

Conjugation

The saccharide conjugates present in the immunogenic compositions of theinvention may be conjugated to a carrier protein using any conjugationtechnique.

In an embodiment, the Streptococcus pneumoniae saccharide is conjugatedto the carrier protein via a linker, for instance a bifunctional linker.The linker is optionally heterobifunctional or homobifunctional, havingfor example a reactive amino group and a reactive carboxylic acid group,two reactive amino groups or two reactive carboxylic acid groups. Thelinker has for example between 4 and 20, 4 and 12, 5 and 10 carbonatoms. A possible linker is adipic acid dihydrazide (ADH). Other linkersinclude B-propionamido (WO 00/10599), nitrophenyl-ethylamine (Geyer etal (1979) Med. Microbiol. Immunol. 165; 171-288), haloalkyl halides(U.S. Pat. No. 4,057,685), glycosidic linkages (U.S. Pat. No. 4,673,574,U.S. Pat. No. 4,808,700), hexane diamine and 6-aminocaproic acid (U.S.Pat. No. 4,459,286). In an embodiment, ADH is used as a linker forconjugating saccharide from serotype 18C.

The saccharide conjugates present in the immunogenic compositions of theinvention may be prepared by any known coupling technique. Theconjugation method may rely on activation of the saccharide with1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form acyanate ester. The activated saccharide may thus be coupled directly orvia a spacer (linker) group to an amino group on the carrier protein.For example, the spacer could be cystamine or cysteamine to give athiolated polysaccharide which could be coupled to the carrier via athioether linkage obtained after reaction with a maleimide-activatedcarrier protein (for example using GMBS) or a haloacetylated carrierprotein (for example using iodoacetimide [e.g. ethyl iodoacetimide HCl]or N-succinimidyl bromoacetate or STAB, or SIA, or SBAP). Optionally,the cyanate ester (optionally made by CDAP chemistry) is coupled withhexane diamine or ADH and the amino-derivatised saccharide is conjugatedto the carrier protein using carbodiimide (e.g. EDAC or EDC) chemistryvia a carboxyl group on the protein carrier. Such conjugates aredescribed in PCT published application WO 93/15760 Uniformed ServicesUniversity and WO 95/08348 and WO 96/29094.

Other suitable techniques use carbodiimides, carbiinides, hydrazides,active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide,S—NHS, EDC, TSTU. Many are described in WO 98/42721. Conjugation mayinvolve a carbonyl linker which may be formed by reaction of a freehydroxyl group of the saccharide with CDI (Bethell et al J. Biol. Chem.1979, 254; 2572-4, Hearn et al J. Chromatogr. 1981. 218; 509-18)followed by reaction with a protein to form a carbamate linkage. Thismay involve reduction of the anomeric terminus to a primary hydroxylgroup, optional protection/deprotection of the primary hydroxyl group’reaction of the primary hydroxyl group with CDI to form a CDI carbamateintermediate and coupling the CDI carbamate intermediate with an aminogroup on a protein.

The conjugates can also be prepared by direct reductive aminationmethods as described in U.S. Pat. No. 4,365,170 (Jennings) and U.S. Pat.No. 4,673,574 (Anderson). Other methods are described in EP-0-161-188,EP-208375 and EP-0-477508.

A further method involves the coupling of a cyanogen bromide (or CDAP)activated saccharide derivatised with adipic acid dihydrazide (ADH) tothe protein carrier by Carbodiimide condensation (Chu C. et al Infect.Immunity, 1983 245 256), for example using EDAC(1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride).

In an embodiment, a hydroxyl group (optionally an activated hydroxylgroup for example a hydroxyl group activated to make a cyanate ester[e.g. using CDAP]) on a saccharide is linked to an amino or carboxylicgroup on a protein either directly or indirectly (through a linker).Where a linker is present, a hydroxyl group on a saccharide isoptionally linked to an amino group on a linker, for example by usingCDAP conjugation. A further amino group in the linker for example ADHmay be conjugated to a carboxylic acid group on a protein, for exampleby using carbodiimide chemistry, for example by using EDAC. In anembodiment, the pneumococcal capsular saccharide(s) is conjugated to thelinker first before the linker is conjugated to the carrier protein.Alternatively the linker may be conjugated to the carrier beforeconjugation to the saccharide.

A combination of techniques may also be used, with somesaccharide-protein conjugates being prepared by CDAP, and some byreductive amination.

In general the following types of chemical groups on a protein carriercan be used for coupling/conjugation:

A) Carboxyl (for instance via aspartic acid or glutamic acid). In oneembodiment this group is linked to amino groups on saccharides directlyor to an amino group on a linker with carbodiimide chemistry e.g. withEDAC.

B) Amino group (for instance via lysine). In one embodiment this groupis linked to carboxyl groups on saccharides directly or to a carboxylgroup on a linker with carbodiimide chemistry e.g. with EDAC. In anotherembodiment this group is linked to hydroxyl groups activated with CDAPor CNBr on saccharides directly or to such groups on a linker; tosaccharides or linkers having an aldehyde group; to saccharides orlinkers having a succinimide ester group.C) Sulphydryl (for instance via cysteine). In one embodiment this groupis linked to a bromo or chloro acetylated saccharide or linker withmaleimide chemistry. In one embodiment this group is activated/modifiedwith bis diazobenzidine.D) Hydroxyl group (for instance via tyrosine). In one embodiment thisgroup is activated/modified with bis diazobenzidine.E) Imidazolyl group (for instance via histidine). In one embodiment thisgroup is activated/modified with bis diazobenzidine.F) Guanidyl group (for instance via arginine).G) Indolyl group (for instance via tryptophan).

On a saccharide, in general the following groups can be used for acoupling: OH, COOH or NH2. Aldehyde groups can be generated afterdifferent treatments known in the art such as: periodate, acidhydrolysis, hydrogen peroxide, etc.

Direct Coupling Approaches:

-   Saccharide-OH+CNBr or CDAP→cyanate ester+NH2-Prot→conjugate-   Saccharide-aldehyde+NH2-Prot→Schiff base+NaCNBH3→conjugate-   Saccharide-COOH+NH2-Prot+EDAC→conjugate-   Saccharide-NH2+COOH-Prot+EDAC→conjugate

Indirect Coupling Via Spacer (Linker) Approaches:

-   Saccharide-OH+CNBr or CDAP→cyanate    ester+NH2—NH2→saccharide-NH2+COOH-Prot+EDAC→conjugate-   Saccharide-OH+CNBr or CDAP→cyanate    ester+NH2—SH→saccharide-SH+SH-Prot (native Protein with an exposed    cysteine or obtained after modification of amino groups of the    protein by SPDP for instance)→saccharide-S—S-Prot-   Saccharide-OH+CNBr or CDAP→cyanate    ester+NH2—SH→saccharide-SH+maleimide-Prot (modification of amino    groups)→conjugate-   Saccharide-OH+CNBr or CDAP→cyanate    ester+NH2—SH→Saccharide-SH+haloacetylated-Prot→Conjugate-   Saccharide-COOH+EDAC+NH2—NH2→saccharide-NH2+EDAC+COOH-Prot→conjugate-   Saccharide-COOH+EDAC+NH2—SH→saccharide-SH+SH-Prot (native Protein    with an exposed cysteine or obtained after modification of amino    groups of the protein by SPDP for instance)→saccharide-S—S-Prot-   Saccharide-COOH+EDAC+NH2—SH→saccharide-SH+maleimide-Prot    (modification of amino groups)→conjugate-   Saccharide-COOH+EDAC+NH2—SH→Saccharide-SH+haloacetylated-Prot→Conjugate-   Saccharide-Aldehyde+NH2—NH2→saccharide-NH2+EDAC+COOH-Prot→conjugate    Note: instead of EDAC above, any suitable carbodiimide may be used.

In summary, the types of protein carrier chemical group that may begenerally used for coupling with a saccharide are amino groups (forinstance on lysine residues), COOH groups (for instance on aspartic andglutamic acid residues) and SH groups (if accessible) (for instance oncysteine residues.

In one embodiment at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 S. pneumoniae saccharides areconjugated to a carrier protein through reductive amination. In oneembodiment less than 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 S. pneumoniae saccharides are conjugatedto a carrier protein through reductive amination. In one embodimentbetween 1 and 23, 2 and 22, 3 and 21, 4 and 20, 5 and 19, 6 and 18, 7and 17, 8 and 16, 9 and 15, 10 and 14, 11 and 13, 1 and 23, and 22, 1and 21, 1 and 20, 1 and 19, 1 and 18, 1 and 17, 1 and 16, 1 and 15, 1and 14, 1 and 13, 1 and 12, 1 and 11, 1 and 10, 1 and 9, 1 and 8, 1 and7, 1 and 6, 1 and 5, 1 and 4, 1 and 3, or 1 or 2 S. pneumoniaesaccharides are conjugated to a carrier protein through reductiveamination. In a further embodiment all of the S. pneumoniae capsularsaccharides are conjugated to a carrier protein through reductiveamination.

In one embodiment at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 S. pneumoniae saccharides areconjugated to a carrier protein through CDAP chemistry. In oneembodiment less than 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 S. pneumoniae saccharides are conjugatedto a carrier protein through CDAP chemistry. In one embodiment between 1and 23, 2 and 22, 3 and 21, 4 and 20, 5 and 19, 6 and 18, 7 and 17, 8and 16, 9 and 15, 10 and 14, 11 and 13, 1 and 23, and 22, 1 and 21, and23, 10 and 22, 10 and 21, 10 and 20, 10 and 19, 10 and 18, 10 and 17, 10and 16, 10 and 15, 10 and 14, 10 and 13, 10 and 12, 10 and 11, 1 and 20,1 and 19, 1 and 18, 1 and 17, 1 and 16, 1 and 15, 1 and 14, 1 and 13, 1and 12, 1 and 11, 1 and 10, 1 and 9, 1 and 8, 1 and 7, 1 and 6, 1 and 5,1 and 4, 1 and 3, or 1 or 2 S. pneumoniae saccharides are conjugated toa carrier protein through CDAP chemistry. In a further embodiment all ofthe S. pneumoniae capsular saccharides are conjugated to a carrierprotein through CDAP chemistry.

In one embodiment the immunogenic composition of the invention comprisesat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21 or 22 saccharides conjugated to a carrier protein throughreductive amination and comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 saccharidesconjugated to a carrier protein through a chemistry other than reductiveamination for example CDAP chemistry.

In an embodiment capsular saccharides from at least one of the serotypesselected from the group consisting of serotypes 1, 3, 19A and 19F areconjugated through a chemistry other than reductive amination and atleast one of the serotypes selected from the group consisting ofserotypes 4, 5, 6A, 6B, 6C, 7F, 9V, 14, 18C and 23F are conjugatedthrough reductive amination. In an embodiment, the immunogeniccomposition of the invention comprises S. pneumoniae capsularsaccharide(s) from serotype 1 or 3 or 19A or 19F; 1 and 3; 1 and 19A; 1and 19F; 3 and 19A; 3 and 19F; 19A and 19F; 1, 3 and 19A; 1, 3 and 19F,1, 19A and 19F; 3, 19A and 19F or 1, 3, 19A and 19F conjugated to aprotein carrier through a chemistry other than reductive animation. Inan embodiment, 19F is conjugated to a carrier protein through achemistry other than reductive amination. In an embodiment, theimmunogenic composition of the invention comprises S. pneumoniaecapsular saccharide from serotype 1 or 3 or 19A or 19F; 1 and 3; 1 and19A; 1 and 19F; 3 and 19A; 3 and 19F; 19A and 19F; 1, 3 and 19A; 1, 3and 19F, 1, 19A and 19F; 3, 19A and 19F or 1, 3, 19A and 19F conjugatedto a protein carrier through cyanylation chemistry such as CDAPchemistry. In an embodiment, 19F is conjugated to a carrier protein byCDAP chemistry. In an embodiment of the invention, the following S.pneumoniae capsular saccharide or saccharides is/are conjugated to acarrier protein by reductive amination; serotype 4, 5, 6A, 6B, 7F, 9V,14, 18C or 23F, 4 and 5, 4 and 6A, 4 and 6B, 4 and 7F, 4 and 9V, 4 and14, 4 and 18C, 4 and 23F, 5 and 6A, 5 and 6B, 5 and 7F, 5 and 9V, 5 and14, 5 and 18C, 5 and 23F, 6A and 6B, 6A and 7F, 6A and 9V, 6A and 14, 6Aand 18C, 6A and 23F, 6B and 7F, 6B and 9V, 6B and 14, 6B and 18C, 6B and23F, 7F and 9V, 7F and 14, 7F and 18C, 7F and 23F, 9V and 14, 9V and18C, 9V and 23F, 14 and 18C, 14 and 23F or 18C and 23F. In anembodiment, serotype 23F is conjugated to a carrier protein by reductiveamination chemistry.

Unconjugated or Conjugated Streptococcus pneumoniae Protein

The immunogenic compositions of the invention may comprise at least oneunconjugated or conjugated S. pneumoniae protein. In one embodiment theat least one unconjugated or conjugated S. pneumoniae protein isselected from the group consisting of Poly Histidine Triad family(PhtX), detoxified pneumolysin (dPly), Choline Binding Protein Family(CbpX), CbpX truncates, LytX (autolytic enzymes) family, LytX truncates,CbpX truncate-LytX truncate chimeric proteins, PcpA (PneumococcalCholine Binding protein A), PspA (Pneumococcal Surface Protein A), PsaA(Pneumococcal Surface Adhesin Protein A), Sp128, Sp101 (Streptococcuspneumoniae 101), Sp130 (Streptococcus pneumoniae 130), SP125(Streptococcus pneumoniae 125) and SP133 (Streptococcus pneumoniae 133).

The Pht (Poly Histidine Triad) family comprises proteins PhtA, PhtB,PhtD, and PhtE. Note that the Pht family can be referred to as eitherthe Poly Histidine Triad family or the Pneumococcal Histidine Triadfamily, thus the terms ‘Poly Histidine Triad’ and ‘PneumococcalHistidine Triad’ can be considered to be interchangeable. The family ischaracterized by a lipidation sequence, two domains separated by aproline-rich region and several histidine triads, possibly involved inmetal or nucleoside binding or enzymatic activity, (3-5) coiled-coilregions, a conserved N-terminus and a heterogeneous C terminus. It ispresent in all strains of pneumococci tested. Homologous proteins havealso been found in other Streptococci and Neisseria. In one embodimentof the invention, the Pht protein of the invention is PhtD. It isunderstood, however, that the terms PhtA, PhtB, PhtD and PhtE refer toproteins having sequences disclosed in the citations below as well asnaturally-occurring (and man-made) variants thereof that have a sequencehomology that is at least 90% identical to the referenced proteins.Optionally it is at least 95% identical or at least 97% identical.

With regards to the PhtX proteins, PhtA is disclosed in WO 98/18930, andis also referred to Sp36. As noted above, it is a protein from the Phtfamily and has the type II signal motif of LXXC. PhtD is disclosed in WO00/37105, and is also referred to Sp036D. As noted above, it also is aprotein from the Pht family and has the type II LXXC signal motif. Inone embodiment the term ‘PhtD’ refers to SEQ ID NO:220. PhtB isdisclosed in WO 00/37105, and is also referred to Sp036B. Another memberof the PhtB family is the C3-Degrading Polypeptide, as disclosed in WO00/17370. This protein also is from the Pht family and has the type IILXXC signal motif. For example, an immunologically functional equivalentis the protein Sp42 disclosed in WO 98/18930. A PhtB truncate(approximately 79 kD) is disclosed in WO99/15675 which is alsoconsidered a member of the Pht family. PhtE is disclosed in WO00/39299and is referred to as BVH-3. Where any Pht protein is referred toherein, it is meant that immunogenic fragments or fusions thereof of thePht protein can be used. For example, a reference to PhtX includesimmunogenic fragments or fusions thereof from any Pht protein. Areference to PhtD or PhtB does not exclude PhtDE (a fusion proteincomprising PhtD and PhtE) or PhtBE (a fusion protein comprising PhtB andPhtE), respectively, as found, for example, in WO0198334.

In one embodiment the at least one unconjugated to conjugatedStreptococccus pneumoniae protein comprises at least one protein fromthe Poly histidine Triad family (for example, the protein may beselected from the group consisting of PhtD, PhtBD and PhtDE fusionprotein). In a further embodiment the at least one unconjugated orconjugated Streptococcus pneumoniae protein is a PhtD protein. In afurther embodiment the PhtD protein comprises an amino acid sequence atleast 85%, 90%, 95%, 98%, 99% or 100% identical to the sequence at aminoacids 21-838 of Sequence ID No. 4 of WO00/37105.

In one embodiment the at least one unconjugated or conjugatedStreptococcus pneumoniae protein is detoxified pneumolysin (dPly). Inone embodiment the pneumolysin has been chemically detoxified. In afurther embodiment the pneumolysin has been chemically detoxified. In ayet further embodiment the pneumolysin has been both chemically andgenetically detoxified.

In a further embodiment the immunogenic composition of the inventioncomprises detoxified pneumolysin (dPly) and PhtD. In a furtherembodiment the immunogenic composition of the invention comprisesunconjugated detoxified pneumolysin (dPly) and unconjugated PhtD.

Concerning the Choline Binding Protein family (CbpX), members of thatfamily were originally identified as pneumococcal proteins that could bepurified by choline-affinity chromatography. The choline bindingproteins are non-covalently bound to phosphorylcholine moieties of cellwall teichoic acid and membrane-associated lipoteichoic acid.Structurally, they have several regions in common over the entirefamily, although the exact nature of the proteins (amino acid sequence,length, etc.) can vary. In general, choline binding proteins comprise anN terminal region (N), conserved repeat regions (R1 and/or R2), aproline rich region (P) and a conserved choline binding region (C), madeup of multiple repeats, that comprises approximately one half of theprotein. As used in this application, the term “Choline Binding Proteinfamily (CbpX)” includes proteins from the group consisting of CholineBinding Proteins as identified in WO97/41151, PbcA, SpsA, PspC, CbpA,CbpD and CbpG. CbpA is disclosed in WO97/41151. CbpD and CbpG aredisclosed in WO00/29434. PspC is disclosed in WO97/09994. PbcA isdisclosed in WO98/21337.5 psA is a choline binding protein disclosed inWO 98/39450. Optionally the Choline Binding Proteins are selected fromthe group consisting of CbpA, PbcA, SpsA and PspC.

An embodiment of the invention comprises CbpX truncates wherein “CbpX”is defined above and “truncates” refers to CbpX proteins lacking 50% ormore of the Choline binding region (C). Optionally such proteins lackthe entire choline binding region. Optionally, the protein truncateslack (i) the choline binding region and (ii) a portion of the N-terminalhalf of the protein as well, yet retain at least one repeat region (R1or R2). Optionally, the truncate retains 2 repeat regions (R1 and R2).Examples of such embodiments are NR1×R2 and R1×R2 as illustrated inWO99/51266 or WO99/51188, however, other choline binding proteinslacking a similar choline binding region are also contemplated withinthe scope of this invention.

The LytX family is membrane associated proteins associated with celllysis. The N-terminal domain comprises choline binding domain(s).However the LytX family does not have all the features found in the CbpXfamily noted above. For the present invention, the LytX family isconsidered distinct from the CbpX family. In contrast with the CbpXfamily, the LytX family C-terminal domain contains the catalytic domainof the LytX protein. The family comprises LytA, LytB and LytC. Withregards to the LytX family, LytA is disclosed in Ronda et al., Eur JBiochem, 164:621-624 (1987). LytB is disclosed in WO 98/18930, and isalso referred to as Sp46. LytC is also disclosed in WO 98/18930, and isalso referred to as Sp91. An embodiment of the invention comprises LytC.

Another embodiment comprises LytX truncates wherein “LytX” is definedabove and “truncates” refers to LytX proteins lacking 50% or more of theCholine binding region. Optionally such proteins lack the entire cholinebinding region. Yet another embodiment of this invention comprises CbpXtruncate-LytX truncate chimeric proteins or fusionproteins. Optionallythe fusion protein comprises NR1×R2 (or R1×R2) of CbpX and theC-terminal portion (Cterm, i.e., the protein without the choline bindingdomains) of LytX (e.g., LytCCterm or Sp91Cterm). Optionally CbpX isselected from the group consisting of CbpA, PbcA, SpsA and PspC.Optionally, it is CbpA. Optionally, LytX is LytC (also referred to asSp91). Another embodiment of the present invention is a PspA or PsaAtruncate lacking the choline binding domain (C) and expressed as afusion protein with LytX. Optionally, LytX is LytC.

With regards to PsaA and PspA, both are discussed in the art. Forexample, PsaA and transmembrane deletion variants thereof have beendescribed by Berry & Paton, Infect Immun 1996 December; 64(12):5255-62.PspA and transmembrane deletion variants thereof have been described in,for example, U.S. Pat. No. 5,804,193, WO 92/14488, and WO 99/53940.

With regards to PcpA, this protein has been described in the art, forexample PcpA has been described in WO2011/075823. The term ‘PcpA’ refersto a protein comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to SEQ ID NO:2 or 7 from WO2011/075823 or fragments of atleast 100, 150, 200, 25 or more consecutive amino acids of SEQ ID NO:2or 7 from WO2011/075823.

Sp128 and Sp130 are disclosed in WO00/76540. Sp125 is an example of apneumococcal surface protein with the Cell Wall Anchored motif of LPXTG(where X is any amino acid). Proteins within this class of pneumococcalsurface protein with this motif has been found to be useful within thecontext of this invention, and is therefore considered a further proteinof the invention. Sp125 itself is disclosed in WO 98/18930, and is alsoknown as ZmpB—a zinc metalloproteinase. Sp101 is disclosed in WO98/06734 (where it has the reference #y85993). It is characterized by aType I signal sequence. Sp133 is disclosed in WO 98/06734 (where it hasthe reference #y85992). It is also characterized by a Type I signalsequence.

Any of these further S. pneumoniae proteins may be present is anunconjugated or conjugated form. One or more S. pneumoniae proteins isoptionally conjugated to an S. pneumoniae saccharide (described in thesection entitled Streptococcus pneumoniae capsular saccharide conjugatesabove). Optionally one or more S. pneumoniae proteins is conjugated to asaccharide from a different bacterium.

The term ‘conjugated to’ in this context means that the protein iscovalently bonded to a saccharide, in this situation the protein isacting as a carrier protein.

In an embodiment the at least one further unconjugated or conjugated S.pneumoniae protein comprises a Poly Histidine family (PhtX) proteinselected from the group consisting of PhtB, PhtE, PhtA, PhtBD and PhtDE.

Adjuvants

In one embodiment the immunogenic composition further comprises anadjuvant.

Suitable adjuvants include, but are not limited to, aluminium salts (forexample, aluminium phosphate or aluminium hydroxide), monophosphoryllipid A (for example 3D-MPL), saponins (for example QS21), oil in wateremulsions, blebs or outer membrane vesicle preparations from Gramnegative bacterial strains (such as those taught by WO02/09746), lipid Aor derivatives thereof, alkyl glucosamide phosphates or combinations oftwo or more of these adjuvants. In one embodiment the adjuvant isaluminium phosphate. In a further embodiment the adjuvant comprises100-750, 150-600, 200-500, 250-450, 300-400, or around 350 μg aluminiumas aluminium phosphate per human dose.

Vaccines

The present invention provides a vaccine comprising the immunogeniccompositions of the invention. Embodiments herein relating to“immunogenic compositions” of the invention are also applicable toembodiments relating to “vaccines” of the invention, and vice versa. Inan embodiment, the vaccine comprises the immunogenic composition of theinvention and a pharmaceutically acceptable excipient.

The vaccines of the invention may be administered by any suitabledelivery route, such as intradermal, mucosal e.g. intranasal, oral,intramuscular or subcutaneous. Other delivery routes are well known inthe art. Vaccine preparation is generally described in Vaccine Design(“The subunit and adjuvant approach” (eds Powell M. F. & Newman M. J.)(1995) Plenum Press New York).

In one aspect, the immunogenic composition of the invention isadministered by the intramuscular delivery route. Intramuscularadministration may be to the thigh or the upper arm. Injection istypically via a needle (e.g. a hypodermic needle), but needle-freeinjection may alternatively be used. A typical intramuscular dose is 0.5ml.

A further aspect of the invention is a method of making a vaccine of theinvention comprising the steps of mixing the unconjugated S. pneumoniaeprotein with the adjuvant composition.

In one aspect of the invention there is provided a method of immunizinga subject against diseases caused by Streptococcus pneumoniae infectioncomprising administering to the subject a therapeutically effective doseof the immunogenic composition or vaccine of the invention. In a furtheraspect of the invention there is provided a method of immunizing asubject against diseases caused by Haemophilus influenzae infectioncomprising administering to the subject a therapeutically effective doseof the immunogenic composition or vaccine of the invention. In a furtherembodiment there is provided a method of immunizing a subject againstdiseases caused by Streptococcus pneumoniae and Haemophilus influenzaeinfection comprising administering to the subject a therapeuticallyeffective dose of the immunogenic composition or vaccine of theinvention. In one embodiment the diseases comprise at least one diseaseselected from the group consisting of pneumonia, invasive pneumococcaldisease (IPD), exacerbations of chronic obstructive pulmonary disease(COPD), otitis media, meningitis, bacteraemia, and conjunctivitis. Inone embodiment the subject is a mammalian subject. In a furtherembodiment the mammalian subject is selected from the group consistingof mouse, guinea pig and human. In one embodiment the subject is anadult human, optionally an elderly human. In a further embodiment thesubject is an infant human.

In a further aspect of the invention is provided an immunogeniccomposition or vaccine of the invention for use in the treatment orprevention of disease caused by Streptococcus pneumoniae infection. In afurther aspect of the invention there is provided an immunogeniccomposition or vaccine of the invention for use in the treatment orprevention of disease caused by Haemophilus influenzae infection. In afurther embodiment there is provided an immunogenic composition orvaccine of the invention for use in the treatment or prevention ofdisease caused by Streptococcus pneumoniae and Haemophilus influenzaeinfection. In one embodiment the use comprises administration of theimmunogenic composition to an adult human host, optionally an elderlyhuman host. In a further embodiment the use comprises administration ofthe immunogenic composition to an infant host. In a further embodimentthe diseases comprise at least one disease selected from the groupconsisting of pneumonia, invasive pneumococcal disease (IPD),exacerbations of chronic obstructive pulmonary disease (COPD), otitismedia, meningitis, bacteraemia, and conjunctivitis.

In a further aspect of the invention there is provided a use of theimmunogenic composition or vaccine of the invention in the manufactureof a medicament for the treatment or prevention of diseases caused byStreptococcus pneumoniae infection. In a further aspect of the inventionthere is provided of use of the immunogenic composition or vaccine ofthe invention in the manufacture of a medicament for the treatment orprevention of diseases caused by Haemophilus influenzae infection. In afurther embodiment there is provided a use of the immunogeniccomposition or vaccine of the invention in the manufacture of amedicament for the treatment or prevention of diseases caused byHaemophilus influenzae and Streptococcus pneumoniae infection. In oneembodiment the diseases comprise at least one disease selected from thegroup consisting of pneumonia, invasive pneumococcal disease (IPD),exacerbations of chronic obstructive pulmonary disease (COPD), otitismedia, meningitis, bacteraemia, and conjunctivitis,

In one embodiment the use comprises administration of the immunogeniccomposition or vaccine of the invention to a host selected from thegroup consisting of an adult human host, an elderly human host and aninfant human host.

Fusion proteins of formula (I) and Streptococcus pneumoniae capsularsaccharide conjugates are useful as immunogens in subjects such asmammals, particularly humans. In particular, the fusion proteins offormula (I) and Streptococcus pneumoniae capsular saccharide conjugatesare useful in inducing an immune response against H. influenzae insubjects, particularly humans. Additionally, the fusion proteins offormula (I) and Streptococcus pneumoniae capsular saccharide conjugatesare useful in inducing an immune response against Streptococcuspneumoniae in subjects, particularly humans. More specifically, thefusion proteins of formula (I) are useful in the treatment or preventionof otitis media and/or AECOPD and/or pneumonia.

In one embodiment, the invention further provides a method for thetreatment or prevention of otitis media in a subject in need thereofcomprising administering to said subject a therapeutically effectiveamount of an immunogenic composition comprising the fusion proteins offormula (I) and Streptococcus pneumoniae capsular saccharide conjugatesas described herein. In another embodiment, the invention provides amethod for the treatment or prevention of acute exacerbations of chronicobstructive pulmonary disease (AECOPD) in a subject in need thereofcomprising administering to said subject a therapeutically effectiveamount of an immunogenic composition comprising the fusion proteins offormula (I) and Streptococcus pneumoniae capsular saccharide conjugatesas described herein.

In another embodiment, the invention provides a method for the treatmentor prevention of pneumonia in a subject in need thereof comprisingadministering to said subject a therapeutically effective amount of animmunogenic composition comprising the fusion proteins of formula (I)and Streptococcus pneumoniae capsular saccharide conjugates as describedherein.

In another embodiment, the invention provides a method for the treatmentor prevention of a H. influenzae infection or disease in a subject inneed thereof, said method comprising administering to said subject atherapeutically effective amount of an immunogenic compositioncomprising the fusion proteins of formula (I) and Streptococcuspneumoniae capsular saccharide conjugates as described herein.

In another embodiment, the invention provides a method for the treatmentor prevention of a S. pneumoniae infection or disease in a subject inneed thereof, said method comprising administering to said subject atherapeutically effective amount of an immunogenic compositioncomprising the fusion proteins of formula (I) and Streptococcuspneumoniae capsular saccharide conjugates as described herein.

Further Definitions

Unless otherwise explained or defined herein, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this disclosurebelongs. For example, definitions of common terms in molecular biologycan be found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. It is further to be understood that all base sizes or aminoacid sizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription. Additionally, numerical limitations given with respect toconcentrations or levels of a substance, such as an antigen may beapproximate. Thus, where a concentration is indicated to be (forexample) approximately 200 pg, it is intended that the concentrationincludes values slightly more or slightly less than (“about” or “˜”) 200pg

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below

The term “comprises” means “includes”. Thus, unless the context requiresotherwise, the word “comprises,” and variations such as “comprise” and“comprising” will be understood to imply the inclusion of a statedcompound or composition (e.g., nucleic acid, polypeptide, antigen) orstep, or group of compounds or steps, but not to the exclusion of anyother compounds, composition, steps, or groups thereof. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

A “subject” as used herein is a mammal, including humans, non-humanprimates, and non-primate mammals such as members of the rodent genus(including but not limited to mice and rats) and members of the orderLagomorpha (including but not limited to rabbits).

As used herein, “adjuvant” means a compound or substance that, whenadministered to a subject in conjunction with a vaccine,immunotherapeutic, or other antigen- or immunogen-containingcomposition, increases or enhances the subject's immune response to theadministered antigen or immunogen (as compared to the immune responsethat would be obtained in the absence of adjuvant). This is to bedistinguished from “adjuvant therapy”, defined by the National CancerInstitute of the United States Institutes of Health in the context ofcancer treatment as additional treatment given after the primarytreatment, to lower the risk that the cancer will recur.

Conservative substitutions are well known and are generally set up asthe default scoring matrices in sequence alignment computer programs.These programs include PAM250 (Dayhoft M. O. et al., (1978), “A model ofevolutionary changes in proteins”, In “Atlas of Protein sequence andstructure” 5(3) M. O. Dayhoft (ed.), 345-352), National BiomedicalResearch Foundation, Washington, and Blosum 62 (Steven Henikoft andJorja G. Henikoft (1992), “Amino acid substitution matrices from proteinblocks”), Proc. Natl. Acad. Sci. USA 89 (Biochemistry): 10915-10919. Theinvention further provides fusion proteins of formula (I) containingconservative amino acid substitutions. For example, the fusion proteinsof formula (I) may contain a conservative substitution of any amino acidfrom PE or PilA of H. influenzae as described in any of the sequencesset forth herein (for example, any PE sequence set forth in SEQ ID NO.4-SEQ ID NO. 57 and/or any PilA sequence set forth in SEQ ID NO. 58-SEQID NO. 121)

As used herein “signal peptide” refers to a short (less than 60 aminoacids, for example, 3 to 60 amino acids) polypeptide present onprecursor proteins (typically at the N terminus), and which is typicallyabsent from the mature protein. The signal peptide (sp) is typicallyrich in hydrophobic amino acids. The signal peptide directs thetransport and/or secretion of the translated protein through themembrane. Signal peptides may also be called targeting signals, transitpeptides, localization signals, or signal sequences. For example, thesignal sequence may be a co-translational or post-translational signalpeptide.

A heterologous signal peptide may be cleaved from a fusion proteinconstruct by signal peptide peptidases during or after proteintransportation or secretion. For example, the signal peptide peptidaseis signal peptide peptidase I. A “heterologous” signal peptide is onewhich is not associated with the protein as it exists in nature.

As used herein “treatment” means the prevention of occurrence ofsymptoms of the condition or disease in a subject, the prevention ofrecurrence of symptoms of the condition or disease in a subject, thedelay of recurrence of symptoms of the condition or disease in asubject, the decrease in severity or frequency of symptoms of thecondition or disease in a subject, slowing or eliminating theprogression of the condition and the partial or total elimination ofsymptoms of the disease or condition in a subject.

As used herein, “optionally” means that the subsequently describedevent(s) may or may not occur, and includes both event(s) that occur andevents that do not occur.

All references or patent applications cited within this patentspecification are incorporated by reference herein.

As used herein ‘infant’ refers to a human 0-2 years old.

As used herein ‘adult’ refers to a human more than 18 years old.

As used herein ‘elderly adult’ refers to a human more than 60 years old,optionally more than 65 years old.

EXAMPLES

In the examples, the following terms have the designated meaning:

-   -   6×his=six histidines;    -   xg=centrifugal force (number gravities)    -   ATP=adenosine triphosphate;    -   BCA=bicinchoninic acid;    -   BSA=bovine serum albumin;    -   ° C.=degrees Celsius;    -   CaCl₂=calcium chloride;    -   CV=column volume;    -   DNA=deoxyribonucleic acid;    -   DSC=differential scanning calorimetry;    -   DTT=dithiothreitol;    -   dNTP=deoxynucleoside triphosphate;    -   EDTA=ethylenediaminetetraacetic acid;    -   FT=flow through;    -   HCl=hydrogen chloride;    -   His=his=histidine;    -   HEPES=4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;    -   IMAC=immobilized metal affinity chromatography;    -   IPTG=isopropyl β-D-1-thiogalactopyranoside;    -   KCl=potassium chloride;    -   K₂HPO₄=dibasic potassium phosphate;    -   KH₂PO₄=monobasic potassium phosphate;    -   LDS=lithium dodecyl sulfate;    -   L=liter;    -   MES=2-(N-morpholino)ethanesulfonic acid;    -   MgCl₂=magnesium chloride;    -   ml=milliliter;    -   RPM=revolutions per minute;    -   min=minute;    -   mM=millimolar;    -   μL=microliter;    -   NaCl=sodium chloride;    -   Na₂HPO₄=dibasic sodium phosphate;    -   NaH₂PO₄=monobasic sodium phosphate;    -   ng=nanogram;    -   nm=nanometer;    -   O/N=overnight;    -   PBS=phosphate buffered saline;    -   PCR=polymerase chain reaction;    -   SB=sample buffer;    -   sec=second;    -   w/v=weight/volume    -   PS=polysaccharide and may be used interchangeably with the term        ‘saccharide’.

1. EXAMPLES Example 1 Fusion Proteins

Fusion proteins were produced with different signal peptides and aminoacid linker sequences. These fusion proteins allowed for secretion ofboth Protein E and PilA (or fragments thereof) without being restrictedto a single bacterial strain. The fusion protein is released into theperiplasm after removal of the heterologous signal peptide by a signalpeptide peptidase. Fusion protein purified from the bacteria does notcontain the heterologous signal peptide. “Purified” proteins are removedfrom the bacteria and lack the signal peptide.

The following table describes fusion protein constructs made.

TABLE 3 Fusion Protein Constructs containing PiIA and Protein E.ConstructID N-terminal-------------------------------------------------------------------------------------C-TerminalLVL312 flgI sp E PiIA fragment G ProtE fragment GGHHHH (A.A.: 40-149 ofSEQ G (A.A.: 18 to 160 of SEQ ID HH ID NO. 58, SEQ ID NO. 4, SEQ ID NO.2 ) NO. 127) A.A. 1 19 21 130 133 275 276 283 LVL291 peIB sp ProtEfragment G PiIA fragment GGHHHH (A.A.: 19 to 160 of SEQ ID G (A.A.:40.149 of HH NO. 4, SEQ ID NO. 124) SEQ ID NO. 58, SEQ ID NO. 127) A.A.1 22 23 164 167 276 277 284 LVL268 peIB sp D ProtE fragment G PiIAfragment GGHHHH (A.A.: 20 to 160 of SEQ ID G (A.A.: 40-149 of HH NO. 4,SEQ ID NO. 125) SEQ ID NO. 58, SEQ ID NO. 127) A.A. 1 22 24 164 167 276277 284 LVL269 nadA sp AT ProtE fragment G PiIA fragment GGHHH ND (A.A.:22 to 160 of SEQ ID G (A.A.: 40-149 of HHH DD NO. 4, SEQ ID NO. 126) SEQID NO. 58, SEQ ID NO. 127) A.A. 1 23 24-29 30 168 171 280 281 288 LVL270M ProtE fragment G PiIA fragment HH (A.A.: 17 to 160 of SEQ ID G (A.A.:40-149 of SEQ HH NO. 4, SEQ ID NO. 122) ID NO. 58, SEQ ID HH NO. 127)A.A. 1 7 8 151 154 263 LVL315 peIB sp M ProtE fragment G PiIA fragmentGGHHHHHH D (A.A.: 22 to 160 of SEQ ID G (A.A.: 40-149 of NO. 4, SEQ IDNO. 126) SEQ ID NO. 58, SEQ ID NO. 127) 1 22 25 163 166 275 276 283LVL317 peIB sp ProtE fragment G PiIA fragment (A.A.: 19 to 160 of SEQ IDG (A.A.: 40-149 of NO. 4, SEQ ID NO. 124) SEQ ID NO. 58, SEQ ID NO. 127)A.A. 1 22 23 164 167 276 LVL318 peIB sp M ProtE fragment G PiIA fragmentD (A.A.: 22 to 160 of SEQ ID G (A.A.: 40-149 of NO. 4, SEQ ID NO. 126)SEQ ID NO. 58, SEQ ID NO. 127) A.A. 1 22 25 163 166 275 LVL702 peIB spProtE fragment G PiIA fragment GGHHHHH (A.A.: 20 to 160 of SEQ ID G(A.A.: 40-149 of H NO. 4, SEQ ID NO. 125) SEQ ID NO. 58, SEQ ID NO 127)A.A. 1 22 23 163 166 275 283 LVL736 peIB sp ProtE fragment G PiIAfragment GG (A.A.: 17 to 160 of SEQ ID NO. G (A.A.: 40-149 of SEQ HH 4,SEQ ID NO. 122) ID NO. 58, SEQ ID HH NO 127) HH A.A. 1 22 23 166 169 278286 LVL737 peIB sp ProtE fragment G PiIA fragment GGH (AA.: 18 to 160 ofSEQ ID G (A.A.: 40-149 of SEQ HHH NO. 4, SEQ ID NO. 123) ID NO. 58, SEQID NO. HH 127) A.A. 1 22 23 165 168 277 285 LVL738 peIB sp ProtEfragment G PiIA fragment GGHHHHHH (A.A.: 22 to 160 of SEQ G (A.A.:40-149 of ID NO. 4, SEQ ID NO. SEQ ID NO. 58, 126) SEQ ID NO. 127) A.A.1 22 23 161 164 273 281 LVL739 peIB sp ProtE fragment G PiIA fragment GGHHRH (A.A.: 23 to 160 of SEQ G (A.A.: 40-149 HH ID NO. 4, SEQ ID NO. ofSEQ ID NO. 179) 58, SEQ ID NO. 127) A.A. 1 22 23 160 163 272 280 LVL740peIB sp ProtE fragment G PiIA fragment GGHHHHH (A.A.: 24 to 160 of G(A.A.: 40-149 H SEQ ID NO. 4, SEQ ID of SEQ ID NO. NO. 180) 58, SEQ IDNO. 127) A.A. 22 23 159 162 271 279 LVL735 peIB sp ProtE fragment G PiIAfragment (A.A.: 20 to 160 of SEQ ID NO. G (A.A.: 40-149 of 4, SEQ ID NO.125) SEQ ID NO. 58, SEQ ID NO. 127) A.A. 1 22 23 163 166 275 LVL778 peIBsp ProtE fragment G PiIA fragment (A.A.: 17 to 160 of SEQ ID NO. G(A.A.: 40-149 of 4, SEQ ID NO. 122) SEQ ID NO. 58, SEQ ID NO. 127) A.A.1 22 23 166 169 278 LVL779 peIB sp ProtE fragment G PiIA fragment (A.A.:18 to 160 of SEQ ID NO. G (A.A.: 40-149 of 4, SEQ ID NO. 123) SEQ ID NO.58, SEQ ID NO. 127) A.A. 1 22 23 165 168 277 LVL780 peIB sp ProtEfragment G PiIA fragment (A.A.: 22 to 160 of SEQ ID NO. G (A .A. : 40149 of 4, SEQ ID NO. 126) SEQ ID NO. 58, SEQ ID NO. 127) A.A. 1 22 23161 164 273 LVL781 peIB sp ProtE fragment G PiIA fragment (A.A.: 23 to160 of SEQ ID NO. G (A.A.: 40-149 of 4, SEQ ID NO. 179) SEQ ID NO. 58,SEQ ID NO. 127) A.A. 1 22 23 160 163 272 LVL782 peIB sp ProtE fragment GPiIA fragment (A.A.: 24 to 160 of SEQ ID NO. G (A.A.: 40-149 of 4, SEQID NO. 180) SEQ ID NO. 58, SEQ ID NO. 127) A.A. 1 22 23 159 162 271 sp =signal peptide; A.A. = amino acid The DNA and amino acid sequences foreach of the signal peptides and plasmids listed in Table 3 are set forthbelow.Signal Sequences:

pelB signal peptide (DNA) - SEQ ID NO. 129:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgc ccagccggcgatggccpelB signal peptide (Amino Acid) - SEQ ID NO. 130:MKYLLPTAAA GLLLLAAQPA MA Flgl signal peptide (DNA) - SEQ ID NO. 131:atgattaaatttctctctgcattaattcttctactggtcacgacggcggc tcaggctFlgl signal peptide (Amino Acid) - SEQ ID NO. 132: MIKFLSALIL LLVTTAAQANadA signal peptide (DNA) - SEQ ID NO. 133:atgaaacactttccatccaaagtactgaccacagccatccttgccacttt ctgtagcggcgcactggcaNadA signal peptide (Amino Acid) - SEQ ID NO. 134:MKHFPSKVLT TAILATFCSG ALAFusion Protein Construct Sequences:

The single underlined portion of the amino acid sequences is from PilAfrom Haemophilus influenzae strain 86-028NP. The embolded underlinedportion of the amino acid sequences was derived from Protein E fromHaemophilus influenza strain 772.

LVL312 (DNA) - SEQ ID NO. 135:atgattaaatttctctctgcattaattcttctactggtcacgacggcggctcaggctgagactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggcgcgcagattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgthatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggccaccaccaccaccaccactaaLVL312 (protein): (flgl sp)(E)(PilA aa 40-149)(GG)(ProtE aa 18-160)(GGHHHHHH) - SEQ ID NO. 136MIKFLSALIL LLVTTAAQAE TKKAAVSELL QASAPYKADV ELCVYSTNET TNCTGGKNGIAADITTAKGY VKSVTTSNGA ITVKGDGTLA NMEYILQATG NAATGVTWTT TCKGTDASLFPANFCGSVTQ GGAQIQKAEQ NDVKLAPPTD VRSGYIRLVK NVNYYIDSES IWVDNQEPQIVHFDAVVNLD KGLYVYPEPK RYARSVRQYK ILNCANYHLT QVRTDFYDEFWGQGLRAAPK KQKKHTLSLT PDTTLYNAAQ IICANYGEAF SVDKKGGHHH HHHLVL291 (DNA) - SEQ ID NO. 137:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggcccagattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttgggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccactaaLVL291 (Protein)(pelB sp)(ProtE aa 19-160)(GG)(PilA aa40-149)(GGHHHHHH) - SEQ ID NO. 138MKYLLPTAAA GLLLLAAQPA MAQIQKAEQN DVKLAPPTDV RSGYIRLVKN VNYYIDSESIWVDNQEPQIV HFDAVVNLDK GLYVYPEPKR YARSVRQYKI LNCANYHLTQVRTDFYDEFW GQGLRAAPKK QKKHTLSLTP DTTLYNAAQI ICANYGEAFS VDKKGGTKKAAVSELLQASA PYKADVELCV YSTNETTNCT GGKNGIAADI TTAKGYVKSV TTSNGAITVKGDGTLANMEY ILQATGNAAT GVTWTTTCKG TDASLFPANF CGSVTQGGHH HHHHLVL268 (DNA) - SEQ ID NO. 139:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgatattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccacLVL268 (protein): (pelB sp)(D)(ProtE aa 20-160)(GG)(PilA aa40-149)(GGHHHHHH) - SEQ ID NO. 140:MKYLLPTAAA GLLLLAAQPA MADIQKAEQN DVKLAPPTDV RSGYIRLVKN VNYYIDSESIWVDNQEPQIV HFDAVVNLDK GLYVYPEPKR YARSVRQYKL LNCANYHLTQVRTDFYDEFW GQGLRAAPKK QKKHTLSLTP DTTLYNAAQI ICANYGEAFS VDKKGGTKKAAVSELLQASA PYKADVELCV YSTNETTNCT GGKNGIAADI TTAKGYVKSV TTSNGAITVKGDGTLANMEY ILQATGNAAT GVTWTTTCKG TDASLFPANF CGSVTQGGHH HHHHLVL269 (DNA) - SEQ ID NO. 141:atgaaacactttccatccaaagtactgaccacagccatccttgccactttctgtagcggcgcactggcagccacaaacgacgacgataaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccactaaLVL269 (protein): (nadA sp)(ATNDDD)(ProtE aa 22-160)(GG)(PilA aa 40-149)(GGHHHHHH) -SEQ ID NO. 142MKHFPSKVLT TAILATFCSG ALAATNDDDK AEQNDVKLAP PTDVRSGYIR LVKNVNYYIDSESIWVDNQE PQIVHFDAVV NLDKGLYVYP EPKRYARSVR QYKILNCANY HLTQVRTDFYDEFWGQGLRA APKKQKKHTL SLTPDTTLYN AAQIICANYG EAFSVDKKGG TKKAAVSELLQASAPYKADV ELCVYSTNET TNCTGGKNGI AADITTAKGY VKSVTTSNGA ITVKGDGTLANMEYILQATG NAATGVTWTT TCKGTDASLF PANFCGSVTQ GGHHHHHHLVL270 (DNA) - SEQ ID NO. 143:atgcaccaccaccaccaccacagcgcgcagattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaataaLVL270 (protein): (MHHHHHH)(ProtE aa 17-160)(GG)(PilA aa40-149) - SEQ ID NO. 144:MHHHHHHSAQ IQKAEQNDVK LAPPTDVRSG YIRLVKNVNY YIDSESIWVD NQEPQINHFDAVVNLDKGLY VYPEPKRYAR SVRQYKILNC ANYHLTQVRT DFYDEFWGQGLRAAPKKQKK HTLSLTPDTT LYNAAQIICA NYGEAFSVDK KGGTKKAAVS ELLQASAPYKADVELCVYST NETTNCTGGK NGIAADITTA KGYVKSVTTS NGAITVKGDG TLANMEYILQATGNAATGVT WTTTCKGTDA SLFPANFCGS VTQ LVL315 (DNA) - SEQ ID NO. 145:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccatggataaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaacattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccactaaLVL315 (protein): (pelB sp)(MD)(ProtE aa 22-160)(GG)(PilA aa40-149)(GGHHHHHH) - SEQID NO. 146:MKYLLPTAAA GLLLLAAQPA MAMDKAEQND VKLAPPTDVR SGYIRLVKNV NYYIDSESIWVDNQEPQIVH FDAVVNLDKG LYVYPEPKRY ARSVRQYKIL NCANYHLTQVRTDFYDEFWG QGLRAAPKKQ KKHTLSLTPD TTLYNAAQII CANYGEAFSV DKKGGTKKAAVSELLQASAP YKADVELCVY STNETTNCTG GKNGIAADIT TAKGYVKSVT TSNGAITVKGDGTLANMEYI LQATGNAATG VTWTTTCKGT DASLFPANCF GSVTQGGHHH HHHLVL317 (DNA) - SEQ ID NO. 147:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggcccatgattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaatatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaatactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaataaLVL317 (protein): (pelB sp)(ProtE aa 19-160)(GG)(PilA aa40-149) - SEQ ID NO. 148:MKYLLPTAAA GLLLLAAQPA MAQIQKAEQN DVKLAPPTDV RSGYIRLVKN VNYYIDSESIWVDNQEPQIV HFDAVVNLDK GLYVYPEPKR YARSVRQYKI LNCANYHLTQVRTDFYDEFW GQGLRAAPKK QKKHTLSLTP DTTLYNAAQI ICANYGEAFS VDKKGGTKKAAVSELLQASA PYKADVELCV YSTNETTNCT GGKNGIAADI TTAKGYVKSV TTSNGAITVKGDGTLANMEY ILQATGNAAT GVTWTTTCKG TDASLFPANF CGSVTQLVL318 (DNA) - SEQ ID NO. 149:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccatggataaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaataaLVL318 (protein): (pelB sp)(MD)(ProtE aa 22-160)(GG)(PilA aa40-149) - SEQ ID NO. 150:MKYLLPTAAA GLLLLAAQPA MAMDKAEQND VKLAPPTDVR SGYIRLVKNV NYYIDSESIWVDNQEPQIVH FDAVVNLDKG LYVYPEPKRY ARSVRQYKIL NCANYHLTQVRTDFYDEFWG QGLRAAPKKQ KKHTLSLTPD TTLYNAAQII CANYGEAFSV DKKGGTKKAAVSELLQASAP YKADVELCVY STNETTNCTG GKNGIAADIT TAKGYVKSVT TSNGAITVKGDGTLANMEYI LQATGNAATG VTWTTTCKGT DASLFPANFC GSVTQLVL702 (DNA) - SEQ ID NO. 181:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccacLVL702 (protein): (pelB sp)(ProtE aa 20-160)(GG)(PilA aa40-149)(GGHHHHHH) - SEQ IDNO. 182:MKYLLPTAAA GLLLLAAQPA MAIQKAEQND VKLAPPTDVR SGYIRLVKNV NYYIDSESIWVDNQEPQIVH FDAVVNLDKG LYVYPEPKRY ARSVRQYKIL NCANYHLTQVRTDFYDEFWG QGLRAAPKKQ KKHTLSLTPD TTLYNAAQII CANYGEAFSV DKKGGTKKAAVSELLQASAP YKADVELCVY STNETTNCTG GKNGIAADIT TAKGYVKSVT TSNGAITVKGDGTLANMEYI LQATGNAATG VTWTTTCKGT DASLFPANFC GSVTQGGHHH HHHLVL736 (DNA) - SEQ ID NO. 183:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccagcgcccagattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccacLVL736 (protein): (pelB sp)(ProtE aa 17-160)(GG)(PilA aa40-149)(GGHHHHHH) - SEQ IDNO. 184:MKYLLPTAAA GLLLLAAQPA MASAQIQKAE QNDVKLAPPT DVRSGYIRLV KNVNYYIDSESIWVDNQEPQ IVHFDAVVNL DKGLYVYPEP KRYARSVRQY KILNCANYHL TQVRTDFYDEFWGQGLRAAP KKQKKHTLSL TPDTTLYNAA QIICANYGEA FSVDKKGGTK KAAVSELLQASAPYKADVEL CVYSTNETTN CTGGKNGIAA DITTAKGYVK SVTTSNGAIT VKGDGTLANMEYILQATGNA ATGVTWTTTC KGTDASLFPA NFCGSVTQGG HHHHHHLVL737 (DNA) - SEQ ID NO. 185:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgcccagattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccacLVL737 (protein): (pelB sp)(ProtE aa 18-160)(GG)(PilA aa40-149)(GGHHHHHH) - SEQ IDNO. 186:MKYLLPTAAA GLLLLAAQPA MAAQIQKAEQ NDVKLAPPTD VRSGYIRLVK NVNYYIDSESIWVDNQEPQI VHFDAVVNLD KGLYVYPEPK RYARSVRQYK ILNCANYHLT QVRTDFYDEFWGQGLRAAPK KQKKHTLSLT PDTTLYNAAQ IICANYGEAF SVDKKGGTKK AAVSELLQASAPYKADVELC VYSTNETTNC TGGKNGIAAD ITTAKGYVKS VTTSNGAITV KGDGTLANMEYILQATGNAA TGVTWTTTCK GTDASLFPAN FCGSVTQGGH HHHHHLVL738 (DNA) - SEQ ID NO. 187:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaatactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccacLVL738 (protein): (pelB sp)(ProtE aa 22-160)(GG)(PilA aa40-149)(GGHHHHHH) - SEQ IDNO. 188:MKYLLPTAAA GLLLLAAQPA MAKAEQNDVK LAPPTDVRSG YIRLVKNVNY YIDSESIWVDNQEPQIVHFD AVVNLDKGLY VYPEPKRYAR SVRQYKILNC ANYHLTQVRTDFYDEFWGQG LRAAPKKQKK HTLSLTPDTT LYNAAQIICA NYGEAFSVDK KGGTKKAAVSELLQASAPYK ADVELCVYST NETTNCTGGK NGIAADITTA KGYVKSVTTS NGAITVKGDGTLANMEYILQ ATGNAATGVT WTTTCKGTDA SLFPANFCGS VTQGGHHHHH HLVL739 (DNA) - SEQ ID NO. 189:ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCGCTGAACAAAATGATGTGAAGCTGGCACCGCCGACTGATGTACGAAGCGGATATATACGTTTGGTAAAGAATGTGAATTATTACATCGATAGTGAATCGATCTGGGTGGATAACCAAGAGCCACAAATTGTACATTTTGATGCAGTGGTGAATTTAGATAAGGGATTGTATGTTTATCCTGAGCCTAAACGTTATGCACGTTCTGTTCGTCAGTATAAGATCTTGAATTGTGCAAATTATCATTTAACTCAAGTACGAACTGATTTCTATGATGAATTTTGGGGACAGGGTTTGCGGGCAGCACCTAAAAAGCAAAAGAAACATACGTTAAGTTTAACACCTGATACAACGCTTTATAATGCTGCTCAGATTATTTGTGCGAACTATGGTGAAGCATTTTCAGTTGATAAAAAAGGCGGCACTAAAAAAGCAGCGGTATCTGAATTACTGCAAGCGTCAGCGCCTTATAAGGCTGATGTGGAATTATGTGTATATAGCACAAATGAAACAACAAACTGTACGGGTGGAAAAAATGGTATTGCAGCAGATATAACCACAGCAAAAGGCTATGTAAAATCAGTGACAACAAGCAACGGTGCAATAACAGTAAAAGGGGATGGCACATTGGCAAATATGGAATATATTTTGCAAGCTACAGGTAATGCTGCAACAGGTGTAACTTGGACAACAACTTGCAAAGGAACGGATGCCTCTTTATTTCCAGCAAATTTTTGCGGAAGTGTCACACAAGGCGGCCACCACCACCACCACCACLVL739 (protein): (pelB sp)(ProtE aa 23-160)(GG)(PilA aa40-149)(GGHHHHHH) - SEQ IDNO. 190:MKYLLPTAAA GLLLLAAQPA MAAEQNDVKL APPTDVRSGY IRLVKNVNYY IDSESIWVDNQEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCA NYHLTQVRTDFYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKK GGTKKAAVSELLQASAPYKA DVELCVYSTN ETTNCTGGKN GIAADITTAK GYVKSVTTSN GAITVKGDGTLANMEYILQA TGNAATGVTW TTTCKGTDAS LFPANFCGSV TQGGHHHHHHLVL740 (DNA) - SEQ ID NO. 191:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccacLVL740 (protein): (pelB sp)(ProtE aa 24-160)(GG)(PilA aa40-149)(GGHHHHHH) - SEQ IDNO. 192:MKYLLPTAAA GLLLLAAQPA MAEQNDVKLA PPTDVRSGYI RLVKNVNYYI DSESIWVDNQEPQIVHFDAV VNLDKGLYVY PEPKRYARSV RQYKILNCAN YHLTQVRTDF YDEFWGQGLRAAPKKQKKHT LSLTPDTTLY NAAQIICANY GEAFSVDKKG GTKKAAVSEL LQASAPYKADVELCVYSTNE TTNCTGGKNG IAADITTAKG YVKSVTTSNG AITVKGDGTL ANMEYILQATGNAATGVTWT TTCKGTDASL FPANFCGSVT QGGHHHHHHLVL735 (DNA) - SEQ ID NO. 193:ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCATTCAGAAGGCTGAACAAAATGATGTGAAGCTGGCACCGCCGACTGATGTACGAAGCGGATATATACGTTTGGTAAAGAATGTGAATTATTACATCGATAGTGAATCGATCTGGGTGGATAACCAAGAGCCACAAATTGTACATTTTGATGCAGTGGTGAATTTAGATAAGGGATTGTATGTTTATCCTGAGCCTAAACGTTATGCACGTTCTGTTCGTCAGTATAAGATCTTGAATTGTGCAAATTATCATTTAACTCAAGTACGAACTGATTTCTATGATGAATTTTGGGGACAGGGTTTGCGGGCAGCACCTAAAAAGCAAAAGAAACATACGTTAAGTTTAACACCTGATACAACGCTTTATAATGCTGCTCAGATTATTTGTGCGAACTATGGTGAAGCATTTTCAGTTGATAAAAAAGGCGGCACTAAAAAAGCAGCGGTATCTGAATTACTGCAAGCGTCAGCGCCTTATAAGGCTGATGTGGAATTATGTGTATATAGCACAAATGAAACAACAAACTGTACGGGTGGAAAAAATGGTATTGCAGCAGATATAACCACAGCAAAAGGCTATGTAAAATCAGTGACAACAAGCAACGGTGCAATAACAGTAAAAGGGGATGGCACATTGGCAAATATGGAATATATTTTGCAAGCTACAGGTAATGCTGCAACAGGTGTAACTTGGACAACAACTTGCAAAGGAACGGATGCCTCTTTATTTCCAGCAAATTTTTGCGGAAGTGTCACACAALVL735 (protein): (pelB sp)(ProtE aa 20-160)(GG)(PilA aa40-149) - SEQ ID NO. 194:MKYLLPTAAA GLLLLAAQPA MAIQKAEQND VKLAPPTDVR SGYIRLVKNV NYYIDSESIWVDNQEPQIVH FDAVVNLDKG LYVYPEPKRY ARSVRQYKIL NCANYHLTQVRTDFYDEFWG QGLRAAPKKQ KKHTLSLTPD TTLYNAAQII CANYGEAFSV DKKGGTKKAAVSELLQASAP YKADVELCVY STNETTNCTG GKNGIAADIT TAKGYVKSVT TSNGAITVKGDGTLANMEYI LQATGNAATG VTWTTTCKGT DASLFPANFC GSVTQLVL778 (DNA) - SEQ ID NO. 195:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccagcgcccagattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagctcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaLVL778 (protein): (pelB sp)(ProtE aa 17-160)(GG)(PilA aa40-149) - SEQ ID NO. 196:MKYLLPTAAA GLLLLAAQPA MASAQIQKAE QNDVKLAPPT DVRSGYIRLV KNVNYYIDSESIWVDNQEPQ IVHFDAVVNL DKGLYVYPEP KRYARSVRQY KILNCANYHL TQVRTDFYDEFWGQGLRAAP KKQKKHTLSL TPDTTLYNAA QIICANYGEA FSVDKKGGTK KAAVSELLQASAPYKADVEL CVYSTNETTN CTGGKNGIAA DITTAKGYVK SVTTSNGAIT VKGDGTLANMEYILQATGNA ATGVTWTTTC KGTDASLFPA NFCGSVTQ LVL779 (DNA) - SEQ ID NO. 197:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgcccagattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaataactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaLVL779 (protein): (pelB sp)(ProtE aa 18-160)(GG)(PilA aa40-149) - SEQ ID NO. 198:MKYLLPTAAA GLLLLAAQPA MAAQIQKAEQ NDVKLAPPTD VRSGYIRLVK NVNYYIDSESIWVDNQEPQI VHFDAVVNLD KGLYVYPEPK RYARSVRQYK ILNCANYHLT QVRTDFYDEFWGQGLRAAPK KQKKHTLSLT PDTTLYNAAQ IICANYGEAF SVDKKGGTKK AAVSELLQASAPYKADVELC VYSTNETTNC TGGKNGIAAD ITTAKGYVKS VTTSNGAITV KGDGTLANMEYILQATGNAA TGVTWTTTCK GTDASLFPAN FCGSVTQ LVL780 (DNA) - SEQ ID NO. 199:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaLVL780 (protein): (pelB sp)(ProtE aa 22-160)(GG)(PilA aa40-149) - SEQ ID NO. 200:MKYLLPTAAA GLLLLAAQPA MAKAEQNDVK LAPPTDVRSG YIRLVKNVNY YIDSESIWVDNQEPQIVHFD AVVNLDKGLY VYPEPKRYAR SVRQYKILNC ANYHLTQVRTDFYDEFWGQG LRAAPKKQKK HTLSLTPDTT LYNAAQIICA NYGEAFSVDK KGGTKKAAVSELLQASAPYK ADVELCVYST NETTNCTGGK NGIAADITTA KGYVKSVTTS NGAITVKGDGTLANMEYILQ ATGNAATGVT WTTTCKGTDA SLFPANFCGS VTQLVL781 (DNA) - SEQ ID NO. 201:AtgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgthatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaLVL781 (protein): (pelB sp)(ProtE aa 23-160)(GG)(PilA aa40-149) - SEQ ID NO. 202:MKYLLPTAAA GLLLLAAQPA MAAEQNDVKL APPTDVRSGY IRLVKNVNYY IDSESIWVDNQEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCA NYHLTQVRTDFYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKK GGTKKAAVSELLQASAPYKA DVELCVYSTN ETTNCTGGKN GIAADITTAK GYVKSVTTSN GAITVKGDGTLANMEYILQA TGNAATGVTW TTTCKGTDAS LFPANFCGSV TQLVL782 (DNA) - SEQ ID NO. 203:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaLVL782 (protein): (pelB sp)(ProtE aa 24-160)(GG)(PilA aa40-149) - SEQ ID NO. 204:MKYLLPTAAA GLLLLAAQPA MAEQNDVKLA PPTDVRSGYI RLVKNVNYYI DSESIWVDNQEPQIVHFDAV VNLDKGLYVY PEPKRYARSV RQYKILNCAN YHLTQVRTDF YDEFWGQGLRAAPKKQKKHT LSLTPDTTLY NAAQIICANY GEAFSVDKKG GTKKAAVSEL LQASAPYKADVELCVYSTNE TTNCTGGKNG IAADITTAKG YVKSVTTSNG AITVKGDGTL ANMEYILQATGNAATGVTWT TTCKGTDASL FPANFCGSVT Q

The full length sequence for PE and PilA from which the above sequenceswere obtained are set forth in SEQ ID NO. 4 (PE) and SEQ ID NO. 58(PilA), respectively.

Example 2 Vector Construction and Transformation

Primers for amplifying PE from H. influenzae strain 772 were designedbased on the sequence of H. influenzae strain Hi Rd. The 5′ primersequence contains one nucleotide difference compared to the NTHi 772sequence, introducing an amino acid difference at position 24 whencompared with the currently reported NTHi 772 genome sequence. Aminoacid #24 in the fusion protein constructs is E (glutamic acid) insteadof K (lysine) as found in NTHi 772.

DNA Sequence for PE from H. influenzae strain Rd. - SEQ ID NO. 151atgaaaaaaattattttaacattatcacttgggttacttaccgcttgttctgctcaaatccaaaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgctgtggtgaatttagataggggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagattttgaattgtgcaaattatcatttaactcaaatacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcaaattatggtaaagcattttcagttgataaaaaataaProtein Sequence for PE from H. influenzae strain Rd. - SEQ ID NO. 152MKKIILTLSL GLLTACSAQI QKAEQNDVKL APPTDVRSGY IRLVKNVNYY IDSESIWVDNQEPQIVHFDA VVNLDRGLYV YPEPKRYARS VRQYKILNCA NYHLTQIRTD FYDEFWGQGLRAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGKAFSVDKKDNA Sequence for PE from H. influenzae strain 772 (as set forth in: Microbes & Infection,Corrigendum to “Identification of a novel Haemophilus influenzae protein important for adhesionto epithelia cells”[Microbes Infect. 10 (2008) 87-97], available online Jul. 6, 2010, “Article inPress”)) - SEQ ID NO. 153atgaaaaaaattattttaacattatcacttgggttacttactgcctgttctgctcaaatccaaaaggctaaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaProtein Sequence for PE from H. influenzae strain 772 (as set forth in: Microbes & Infection,Corrigendum to “Identification of a novel Haemophilus influenzae protein important for adhesionto epithelia cells”[Microbes Infect. 10 (2008) 87-97], available online Jul. 6, 2010, “Article inPress”)) - SEQ ID NO. 154MKKIILTLSL GLLTACSAQI QKAKQNDVKL APPTDVRSGY IRLVKNVNYY IDSESIWVDNQEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCA NYHLTQVRTD FYDEFWGQGLRAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKKVector Construction:

To generate LVL312, LVL291, LVL268, LVL269, LVL270, LVL702, LVL735,LVL778, LVL779, LVL780, LVL781 and LVL782, a polymerase chain reaction(PCR) preparation of the following components was prepared (specificcomponents are subsequently exemplified): 36.6 μl of deionized water, 5μl of buffer #1 10×, 5 μl of dNTPs 2 mM, 2 μl MgCl₂ 25 mM, 0.4 μl ofprimer #1 (50 μM), 0.4 μl of primer #2 (50 μM), 0.5 μl of template (100ng/μl) and 0.4 μl of KOD HiFi DNA polymerase 2.5 units/μl (NOVAGEN®) wasformulated. Polymerase chain reaction involved 25 cycles of 15 secondsof denaturation at 98° C., 2 seconds for annealing at 55° C. and 20seconds of primer extension at 72° C. The PCR products were purifiedusing QIAQUICK® PCR purification kit (QIAGEN®). This product was usedunder conditions recommended by the supplier which were: the addition of5 volumes Buffer PB, provided in the QIAQUICK® PCR purification kit, to1 volume of the PCR preparation. The PCR preparation with Buffer PB wassubsequently mixed by vortex. A QIAQUICK® column was placed into a 2 mlcollection tube. To bind DNA in the PCR preparation to the column, themixed sample was applied to the QIAQUICK® column and centrifuged for30-60 seconds at 14 000 RPM. The flow-through was discarded and theQIAQUICK® column was placed back in the same tube. To wash the bound DNA0.75 ml Buffer PE, provided in the QIAQUICK® PCR purification kit, wasadded to the QIAQUICK® column, and the column was centrifuged for 30-60seconds at 14 000 RPM. The flow-through was discarded and the QIAQUICK®column was placed back in the same tube. The QIAQUICK® column wascentrifuged once more in the 2 ml collection tube for 1 minute to removeresidual wash buffer. Each QIAQUICK® column was placed in a clean 1.5 mlmicrocentrifuge tube. To elute the DNA, 33 μl water was added to thecenter of the QIAQUICK® membrane and the column was centrifuged for 1minute at 14 000 RPM. Restriction enzymes and buffer related wereobtained from New England BioLabs. For example, approximately 5 μl ofpET26b vector (100 ng/μl), 2 μl of NEBuffer 2 (New England Biolabs,1×NEBuffer 2: 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl₂, 1 mMdithiothreitol, pH 7.9 at 25° C.), 1 μl of NdeI (20 000 units/ml), 1 μlof HindIII (20 000 units/ml) and 11 μl of deionized water were mixed andincubated for two hours at 37° C. for DNA digestion. Thereafter, asecond step of purification was performed using the QIAQUICK® PCRpurification kit (QIAGEN®) with the procedure described above.

Ligation was performed using Quick T4 DNA ligase and Quick LigationReaction Buffer from New England BioLabs. For example, around 10 ng ofvector and 30 ng of insert in 10 μl of deionized water were mixed with10 μl of 2× Quick Ligation Reaction Buffer (New England Biolabs, 132 mMTris-HCl, 20 mM MgCl₂, 2 mM dithiothreitol, 2 mM ATP, 15% polyethyleneglycol, pH 7.6 at 25° C.) and 1 μl of Quick T4 DNA ligase (New EnglandBiolabs). The enzymatic reaction was incubated for 5 minutes at roomtemperature before transformation.

To generate LVL315, LVL317, LVL318, LVL736, LVL737, LVL738, LVL739 andLVL740, a PCR preparation of the following components was prepared: 40μl of deionized water, 5 μl of reaction buffer 10×, 1 μl of dNTPs mix, 1μl of primer #1 (10 μM), 1 μl of primer #2 (10 μM), 1 μl of template (25ng/μl) and 1 μl of PfuUltra High-Fidelity DNA polymerase 2.5 units/μl(QuikChange II Site-Directed Mutagenesis Kit, Agilent Technologies,Stratagene Division) was formulated. Polymerase chain reaction involvedone cycle of denaturation at 95° C. for 30 sec, 18 cycles of 30 sec ofdenaturation at 95° C., 1 min for annealing at 55° C. and 5 min 30 secof primer extension at 68° C. The PCR products were digested using 1 μlof Dpnl restriction enzyme at 37° C. for one hour before transformation.

A detailed list of PCR primer sequences used for amplifications isillustrated in Table 4.

To generate pRIT16711, the PE gene fragment coding for amino acids 22 to160 of SEQ ID NO. 4, which excludes the sequence coding for itscorresponding secretion signal, was amplified by PCR from genomic DNA ofNTHi strain 772. The amplification primers were designed based on theavailable strain Hi Rd sequence (at that time, the 772 sequence was notknown). The 5′ primer sequence contains one mutation compared to theNTHi 772 sequence (sequence as now available), introducing one aminoacid difference in PE coding sequence at position 24, glutamic acid (E)instead of lysine (K). After PCR amplification, the insert was cloned inthe pET-26(+) expression vector (NOVAGEN®) using BamHI and XhoIrestriction sites.

To generate pRIT16671, a DNA fragment coding for a PilA gene fragment(amino acids 40 to 149 of SEQ ID NO. 58, SEQ ID NO. 127), which excludesits leader peptide as well as a portion of the predicted hydrophobicalpha helix, was amplified from genomic DNA of NTHi strain 86-028NP andcloned into the pET15 expression vector. The vector pRIT16790(containing amino acids 40 to 149 from NTHi strain 86-028NP) was used asa template to generate the vector pRIT16671. The PilA gene fragment wasamplified by PCR using the vector pRIT16790 and primers MDES PILA-3 andMDES PILA-4. The PilA fragment was cloned into the pET-26 expressionvector using NdeI/XhoI restriction sites. The DNA sequence encoding sixhistidine (his) amino acids was incorporated into the 5′ primer to addsix histidines (6×his) at the N-terminal end of the PilA sequence (MDESPILA-3).

To generate LVL312 (FlgI signal peptide-E-PilA fragment-GG-PEfragment-GGHHHHHH), a polymerase chain reaction was performed to amplifythe PilA gene (amino acids 40-149/strain 86-028NP) using the pRIT16671vector as a template and primers CAN534 and CAN537. DNA sequencecorresponding to FlgI signal peptide (sp) and glutamic acid (E) aminoacid was incorporated into the 5′ primer (CAN534). To link the PilAsequence to PE sequence, DNA sequence corresponding to the two glycine(GG) amino acids linker and the N-terminal PE amino acids wereincorporated into the 3′ primer (CAN537). Another polymerase chainreaction was performed to amplify the PE gene (amino acids 18-160) usingpRIT16711 vector as a template and primers CAN536 and CAN538. DNAsequence corresponding to the C-terminal PilA amino acids and GG aminoacids were incorporated into the 5′ primer to link pilA to PE sequence(CAN536). DNA sequence corresponding to the GG amino acids linker and6×his amino acids were incorporated into the 3′ primer (CAN538).Finally, to generate LVL312, a third polymerase chain reaction wasperformed to amplify the PilA and PE genes in fusion with the FlgIsignal peptide at the N-terminus, a glutamic acid (E) amino acid betweenFlgI and pilA, a GG linker between PilA and PE sequences and a GG linkerbetween PE and the 6×his amino acids at the C-terminus. To achieve thisamplification, the products of the two polymerase chain reactionsdescribed above were used as a template with primers CAN534 and CAN538.DNA sequence corresponding to NdeI restriction site was incorporatedinto the 5′ primer and HindIII restriction site was incorporated intothe 3′ primer. The generated PCR product was then inserted into thepET-26b(+) cloning vector (NOVAGEN®).

To generate LVL291 (pelB signal peptide-PE fragment-GG-PilAfragment-GG-6×his), a polymerase chain reaction was performed to amplifythe PE gene (amino acids 19-160) using the pRIT16711 vector as atemplate and primers CAN544 and CAN546. DNA sequence corresponding topelB signal peptide (sp) amino acids was incorporated into the 5′ primer(CAN544). To link the PilA sequence to the PE sequence, DNA sequencecorresponding to GG amino acids linker and the N-terminal PilA aminoacids were incorporated into the 3′ primer (CAN546). Another polymerasechain reaction was performed to amplify the PilA gene (amino acids40-149 of SEQ ID NO. 58, SEQ ID NO. 127) using the pRIT16671 vector as atemplate with primers CAN545 and CAN535. DNA sequence corresponding tothe C-terminal PE amino acids and GG amino acids were incorporated intothe 5′ primer (CAN545) to link the PilA sequence to the PE sequence. DNAsequence corresponding to linker GG amino acids and 6×his amino acidswere incorporated into the 3′ primer (CAN535). Finally, to generateLVL291, a third polymerase chain reaction was performed to amplify thePE and PilA genes in fusion with the pelB signal peptide at theN-terminus, a GG linker between the PE and PilA sequences and a GGlinker between PilA and 6×his amino acids at the C-terminus. To achievethis amplification, the products of two polymerase chain reactionsdescribed above were used as a template with primers CAN544 and CAN535.DNA sequence corresponding to NdeI restriction site was incorporatedinto the 5′ primer and HindIII restriction site was incorporated intothe 3′ primer. The generated PCR product was then inserted into thepET-26b(+) cloning vector (NOVAGEN®).

To generate LVL268 (pelB signal peptide-D-PE fragment-GG-PilAfragment-GG-6×his), a polymerase chain reaction was performed to amplifythe PE gene (amino acids 20-160) using the pRIT16711 vector as atemplate with primers CAN547 and CAN546. DNA sequence corresponding tothe pelB signal peptide (sp) amino acids and aspartic acid (D) aminoacid were incorporated into the 5′ primer (CAN547). To link the PilAsequence to the PE sequence, DNA sequence corresponding to GG aminoacids linker and the N-terminal PilA amino acids were incorporated intothe 3′ primer (CAN546). Another polymerase chain reaction was performedto amplify the PilA gene (amino acids 40-149/NTHi strain 86-028NP) usingthe pRIT16671 vector as a template with CAN545 and CAN535. DNA sequencecorresponding to the C-terminal PE amino acids and GG amino acids wereincorporated into the 5′ primer (CAN545) to link the PilA sequence tothe PE sequence. DNA sequence corresponding to linker GG amino acids and6×his amino acids were incorporated into the 3′ primer (CAN535).Finally, to generate LVL268, a third polymerase chain reaction wasperformed to amplify the PE and PilA genes in fusion with the pelBsignal peptide at the N-terminus, a D amino acid between pelB signalpeptide and PE, a GG linker between PE and pilA sequences and a GGlinker between PilA and 6×his amino acids in C-term. To achieve thisamplification, the products of the two polymerase chain reactionsdescribed above were used as a template with primers CAN547 and CAN535.DNA sequence corresponding to NdeI restriction site was incorporatedinto the 5′ primer and HindIII restriction site was incorporated intothe 3′ primer. The generated PCR product was then inserted into thepET-26b(+) cloning vector (NOVAGEN®).

To generate LVL269 (NadA signal peptide-ATNDDD-PE fragment-GG-PilAfragment-GG-6×his), a polymerase chain reaction was performed to amplifythe PE gene (amino acids 22-160 of SEQ ID NO. 4) using the pRIT16711vector as a template with primers CAN548 and CAN546. DNA sequencecorresponding to pelB signal peptide (sp) amino acids and ATNDDD aminoacids were incorporated into the 5′ primer (CAN548). To link the PilAsequence to the PE sequence, DNA sequence corresponding to the GG aminoacids linker and the N-terminal PilA amino acids were incorporated intothe 3′ primer (CAN546). Another polymerase chain reaction was performedto amplify the PilA gene (amino acids 40-149 of SEQ ID NO. 58, SEQ IDNO. 127) using the pRIT16671 vector as a template with primers CAN545and CAN535. DNA sequence corresponding to the C-terminal PE amino acidsand GG amino acids were incorporated into the 5′ primer to link the PilAsequence to the PE sequence (CAN545). DNA sequence corresponding tolinker GG amino acids and 6×his amino acids were incorporated into the3′ primer (CAN535). Finally, to generate LVL269, a third polymerasechain reaction was performed to amplify the PE and PilA gene in fusionwith the NadA signal peptide at the N-terminus, ATNDDD amino acidsbetween the pelB signal peptide and PE, a GG linker between the PE andpilA sequences and a GG linker between PilA and 6×his amino acids at theC-terminus. To achieve this amplification, the products of the twopolymerase chain reactions describe above were used as a template withprimers CAN548 and CAN535. DNA sequence corresponding to NdeIrestriction site was incorporated into the 5′ primer and HindIIIrestriction site was incorporated into the 3′ primer. The generated PCRproduct was then inserted into the pET-26b(+) cloning vector (NOVAGEN®).

To generate LVL270 (M-6×His-PE fragment-GG-PilA fragment), a polymerasechain reaction was performed to amplify the PE gene (amino acids 17-160)using the pRIT16711 vector as a template with primers CAN540 and CAN542.DNA sequence corresponding to 6×his amino acids were incorporated intothe 5′ primer (CAN540). To link the PilA sequence to the PE sequence,DNA sequence corresponding to the GG amino acids linker and theN-terminal PilA amino acids were incorporated into the 3′ primer(CAN542). Another polymerase chain reaction was performed to amplify thePilA gene (amino acids 40-149/NTHi strain 86-028NP) using pRIT16671vector as a template with primers CAN541 and CAN543. DNA sequencecorresponding to the C-terminal PE amino acids and GG amino acids wereincorporated into the 5′ primer (CAN541) to link the PilA to the PEsequence. Finally, to generate LVL270, a third polymerase chain reactionwas performed to amplify the 6-his-PE-GG-PilA gene in fusion. To achievethis amplification, the products of the two polymerase chain reactionsdescribe above were used as a template with primers CAN540 and CAN543.DNA sequence corresponding to NdeI restriction site was incorporatedinto the 5′ primer and HindIII restriction site was incorporated intothe 3′ primer. The generated PCR product was then inserted into thepET-26b(+) cloning vector (NOVAGEN®).

To generate LVL315 (pelB signal peptide-MD-PE fragment-GG-PilAfragment-GG-6×his), a site-directed mutagenesis was performed to changethe N-terminal PE amino acid sequence from QIQ to MD using LVL291 as atemplate with primers CAN670 and CAN671 and the QuikChange IISite-Directed Mutagenesis Kit (Agilent Technologies, StratageneDivision).

To generate LVL317 (pelB signal peptide-PE fragment-GG-pilA fragment), asite-directed mutagenesis was performed to incorporate a stop codonbetween the PilA gene and the DNA sequence corresponding to GGHHHHHHamino acid residues (SEQ ID NO: 3) using LVL291 as a template withprimers CAN678 and CAN679 and the QuikChange II Site-DirectedMutagenesis Kit (Agilent Technologies, Stratagene Division).

To generate LVL318 (pelB signal peptide-MD-PE-GG-PilA), a site-directedmutagenesis was performed to incorporate a stop codon between the PilAgene and the DNA sequence corresponding to GGHHHHHH amino acid residues(SEQ ID NO: 3) using LVL315 as a template with primers CAN678 and CAN679and the QuikChange II Site-Directed Mutagenesis Kit (AgilentTechnologies, Stratagene Division).

To generate LVL702 (LVL291 ΔQ), a polymerase chain reaction wasperformed using the LVL291 vector as template and primers CAN1517 andCAN1518. Deletion of three nucleotides corresponding to the amino acid Qat the position 23 on LVL291 sequence was incorporated to the 5′ primer.The only difference between LVL702 and LVL291 is the deletion of aminoacid Q at the position 23 on LVL291 sequence. NdeI and HindIIIrestriction sites were incorporated into the 5′ and 3′ primersrespectively. The generated PCR product was then inserted into thepET-26b(+) cloning vector (NOVAGEN®).

To generate LVL735 (LVL317 ΔQ), a polymerase chain reaction wasperformed using the LVL317 vector as template and primers CAN1517 andCAN1519. Deletion of three nucleotides corresponding to the amino acid Qat the position 23 on LVL317 sequence was incorporated to the 5′ primer.The only difference between LVL735 and LVL317 is the deletion of aminoacid Q at the position 23 on LVL317 sequence. NdeI and HindIIIrestriction sites were incorporated into the 5′ and 3′ primersrespectively. The generated PCR product was then inserted into thepET-26b(+) cloning vector (NOVAGEN®).

To generate LVL736 (LVL291+SA), a site-directed mutagenesis wasperformed to add amino acids S and A between amino acid 22 and 23 onLVL291 sequence. LVL291 was used as template with primers CAN1531 andCAN1532 and the QuikChange II Site-Directed Mutagenesis Kit (AgilentTechnologies, Stratagene Division).

To generate LVL737 (LVL291+A), a site-directed mutagenesis was performedto add amino acid A between amino acid 22 and 23 on LVL291 sequence.LVL291 was used as template with primers CAN1529 and CAN1530 and theQuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies,Stratagene Division).

To generate LVL738 (LVL291 ΔQIQ), a site-directed mutagenesis wasperformed to delete amino acids Q, I and Q at positions 23 to 25 onLVL291 sequence. LVL291 was used as template with primers CAN1523 andCAN1524 and the QuikChange II Site-Directed Mutagenesis Kit (AgilentTechnologies, Stratagene Division).

To generate LVL739 (LVL291 ΔQIQK), a site-directed mutagenesis wasperformed to delete amino acids Q, I, Q and K at positions 23 to 26 onLVL291 sequence. LVL291 was used as template with primers CAN1525 andCAN1526 and the QuikChange II Site-Directed Mutagenesis Kit (AgilentTechnologies, Stratagene Division).

To generate LVL740 (LVL291 ΔQIQKA), a site-directed mutagenesis wasperformed to delete amino acids Q, I, Q, K and A at positions 23 to 27on LVL291 sequence. LVL291 was used as template with primers CAN1527 andCAN1528 and the QuikChange II Site-Directed Mutagenesis Kit (AgilentTechnologies, Stratagene Division).

To generate LVL778 (LVL736 Δ6×His tag), LVL779 (LVL737 Δ6×His tag),LVL780 (LVL738 Δ6×His tag), LVL781 (LVL739 Δ6×His tag) and LVL782(LVL740 Δ6×His tag) a polymerase chain reaction was performed using theLVL736, LVL737, LVL738, LVL739 and LVL740 vectors as template,respectively, with primers CAN1669 and CAN543. Deletion of 6×His tagcorresponds to the amino acid sequence GGHHHHHH (SEQ ID NO. 3) at theC-terminal sequences. This deletion was incorporated to the 3′ primer.NdeI and HindIII restriction sites were incorporated into the 5′ and 3′primers respectively. The generated PCR product was then inserted intothe pET-26b(+) cloning vector (NOVAGEN®).

TABLE 4PCR primer sequences used for PE, PilA and PE-PilA amplificationsDNA Sequence Primer ID 5′-3′ CAN534CACACACATATGATTAAATTTCTCTCTGCATTAATTCTTCTACTGGTCACGACGGCGGCTCAGGCTGAGACTAAAAAAGCAGCGGTATCTG (SEQ ID NO. 155) CAN535TGTGTGAAGCTTTTAGTGGTGGTGGTGGTGGTGGCCGCCTTGTGTGACACTTCCGCAAAAATTTGC (SEQ ID NO. 156) CAN536TTTGCGGAAGTGTCACACAAGGCGGCGCGCAGATTCAGAAGGCTGAACAAAATGATGT (SEQ ID NO. 157) CAN537ACATCATTTTGTTCAGCCTTCTGAATCTGCGCGCCGCCTTGTGTGACACTTCCGCAAA (SEQ ID NO. 158) CAN538TGTGTGAAGCTTTTAGTGGTGGTGGTGGTGGTGGCCGCCTTTTTTATCAACTGAAAATG (SEQ ID NO. 159) CAN540CACACACATATGCACCACCACCACCACCACAGCGCGCAGATTCAGAAGGCTGAACAAAATGATGT (SEQ ID NO. 160) CAN541CATTTTCAGTTGATAAAAAAGGCGGCACTAAAAAAGCAGCGGTATC (SEQ ID NO. 161) CAN542GATACCGCTGCTTTTTTAGTGCCGCCTTTTTTATCAACTGAAAATG (SEQ ID NO. 162) CAN543TGTGTGAAGCTTTTATTGTGTGACACTTCCGCAAA (SEQ ID NO. 163) CAN544CACACACATATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCCAGATTCAGAAGGCTGAACAAAATGATG T (SEQ ID NO. 164)CAN545 GCATTTTCAGTTGATAAAAAAGGCGGCACTAAAAAAGCAGCGGTATCTG(SEQ ID NO. 165) CAN546CAGATACCGCTGCTTTTTTAGTGCCGCCTTTTTTATCAACTGAAAATGC (SEQ ID NO. 166)CAN547 CACACACATATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCGATATTCAGAAGGCTGAACAAAATGATG T (SEQ ID NO. 167)CAN548 CACACACATATGAAACACTTTCCATCCAAAGTACTGACCACAGCCATCCTTGCCACTTTCTGTAGCGGCGCACTGGCAGCCACAAACGACGACGATAAGGCTGAACAAAATGATG (SEQ ID NO. 168) CAN670GCCGGCGATGGCCATGGATAAGGCTGAACAAAATG (SEQ ID NO. 169) CAN671CATTTTGTTCAGCCTTATCCATGGCCATCGCCGGC (SEQ ID NO. 170) CAN678GGAAGTGTCACACAATAAGGCGGCCACCACCACC (SEQ ID NO. 171) CAN679GGTGGTGGTGGCCGCCTTATTGTGTGACACTTCC (SEQ ID NO. 172) CAN1517GATATACATATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCATTCAGAAGGCTGAACAAAA (SEQ ID NO. 205) CAN1518GGCCGCAAGCTTTTAGTGGTGGTGGTGGTGGTGGCCGCC (SEQ ID NO. 206) CAN1519GGCCGCAAGCTTTTATTGTGTGACACTTCC (SEQ ID NO. 207) CAN1523GCTGCCCAGCCGGCGATGGCCAAGGCTGAACAAAATGATGTG (SEQ ID NO. 208) CAN1524CACATCATTTTGTTCAGCCTTGGCCATCGCCGGCTGGGCAGC (SEQ ID NO. 209) CAN1525GCTGCCCAGCCGGCGATGGCCGCTGAACAAAATGATGTGAAGC (SEQ ID NO. 210) CAN1526GCTTCACATCATTTTGTTCAGCGGCCATCGCCGGCTGGGCAGC (SEQ ID NO. 211) CAN1527GCTGCCCAGCCGGCGATGGCCGAACAAAATGATGTGAAGCTGG (SEQ ID NO. 212) CAN1528CCAGCTTCACATCATTTTGTTCGGCCATCGCCGGCTGGGCAGC (SEQ ID NO. 213) CAN1529GCTGCCCAGCCGGCGATGGCCGCCCAGATTCAGAAGGCTGAAC (SEQ ID NO. 214) CAN1530GTTCAGCCTTCTGAATCTGGGCGGCCATCGCCGGCTGGGCAGC (SEQ ID NO. 215) CAN1531GCTGCCCAGCCGGCGATGGCCAGCGCCCAGATTCAGAAGGCTGAAC (SEQ ID NO. 216) CAN1532GTTCAGCCTTCTGAATCTGGGCGCTGGCCATCGCCGGCTGGGCAGC (SEQ ID NO. 217) CAN1669CACACACATATGAAATACCTGCTGCCGACC (SEQ ID NO. 218) MDesPILA-3GAATTCCATATGCACCATCACCATCACCATACTAAAAAAGCAGCGGTATCTGA A (SEQ ID NO. 173)MDesPILA-4 GCGCCGCTCGAGTCATTGTGTGACACTTCCGC (SEQ ID NO. 174) MnoNTHi-44GCCCAGCCGGCGATGGCCCAGATCCAGAAGGCTGAACAAAATG (SEQ ID NO. 175) MnoNTHi-45CATTTTGTTCAGCCTTCTGGATCTGGGCCATCGCCGGCTGGGC (SEQ ID NO. 176)Transformation

Escherichia coli BLR (DE3) or E. coli HMS (DE3) cells were transformedwith plasmid DNA according to standard methods with CaCl₂-treated cells.(Hanahan D. <<Plasmid transformation by Simanis.>> In Glover, D. M.(Ed), DNA cloning. IRL Press London. (1985): p. 109-135.). Briefly, BLR(DE3) or HMS174(DE3) competent cells were gently thawed on ice.Approximately 4 μl of plasmid (10-100 ng) were mixed using 50-100 μlcompetent cells. Thereafter, this formulation was incubated on ice for30 min. To perform the transformation reaction, the formulation was heatpulsed at 42° C. for 45 seconds then incubated on ice for 2 minutes.Approximately 0.5 ml of SOC medium (Super Optimal broth with Cataboliterepression) was added to the transformed cells and the cell culture wasincubated at 37° C. for one hour before plating on Luria-Bertani (LB)agar with 50 ug/ml kanamycin. Around 100 μl of transformed cell culturewas plated and incubated overnight at 37° C.

BLR (DE3): BLR is a recA⁻ derivative of BL21 (F-ompT hsdSB(rB-mB-) galdcm (DE3). This E. coli strain used for expression of recombinantproteins improves plasmid monomer yields and may help stabilize targetplasmids containing repetitive sequences or whose products may cause theloss of the DE3 prophage. (Studier, F. W. (1991) J. Mol. Biol. 219:37-44). The detailed genotype of E. coli BLR (DE3) has been published byNOVAGEN®. (F-ompT hsdSB (rB-mB-) gal dcm Δ(srl-recA)306::Tn10 (TetR)(DE3).

HMS174 (DE3): HMS174 strains provide the recA mutation in a K-12background. Like BLR, these strains may stabilize certain target geneswhose products may cause the loss of the DE3 prophage. The detailedgenotype of E. coli HMS174 (DE3) has been published by NOVAGEN®.(F-recA1 hsdR(rK12−mK12+) (DE3) (Rif R).

Production Using BLR (DE3) and Characterization of His Tagged Constructsare Described in Example 3 Through Example 6

Example 3 Protein Expression Using Shake Flask

Generally, one confluent agar plate inoculated with Escherichia coli BLR(DE3) transformed with recombinant plasmid was stripped, resuspended inculture media and used to inoculate 800 ml of LB broth (Becton,Dickinson and Company)±1% (weight/volume, w/v) glucose (Laboratoire MAT,catalogue number: GR-0101) and 50 μg/ml kanamycin (Sigma) to obtainO.D._(600 nm) between 0.1 and 0.2. Cultures were incubated at 37° C.with agitation of 250 RPM to reach an O.D._(600 nm) of ˜0.8.

One ml of each culture was then collected, centrifuged at 14 000 RPM for5 minutes and supernatants and pellets were frozen at −20° C.separately.

At an O.D._(600 nm)˜0.8, the BLR (DE3) cultures were cooled down (−20°C., 20 minutes or 4° C., 1 hour, preferably at 4° C. for 1 hour) beforeinducing the expression of the recombinant protein by addition of 1 mMisopropyl β-D-1-thiogalactopyranoside (IPTG; EMD Chemicals Inc.,catalogue number: 5815) and incubation overnight at 16, 22 and 30° C.,or 3 hours at 37° C. with agitation of 250 RPM, preferably overnight at22° C. After the induction period the cultures were centrifuged at 14000 RPM for 5 minutes or 6 000 RPM for 15 minutes and supernatant (mediafraction sample) and pellets (containing soluble and insolublefractions) were frozen at −20° C. separately.

These conditions are used for periplasmic protein expression.

Example 4 Protein Purification Using Shake Flask, Cell Pastes, HisTagged Constructs

Each bacterial pellet obtained after induction was resuspended in 20 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH8.0) containing 500 mM NaCl, 10 mM imidazole and Roche COMPLETE®Protease Inhibitor Cocktail (1 tablet/50 ml of HEPES buffer containing500 mM NaCl, Roche COMPLETE® ULTRA tablets, Roche DiagnosticsCorporation).

Alternatively, 20 to 50 mM bicine buffer may be used instead of HEPESbuffer containing NaCl. For example, 20 mM bicine buffer may be used.Bacteria were lysed using a Constant System 1.1 KW 2×30 000 PSI (poundsper square inch). Soluble (supernatant) and insoluble (pellet)components were separated by centrifugation at 20 000 g for 20 min at 4°C.

6-His tagged-proteins were purified under native conditions onimmobilized metal affinity chromatography (IMAC) using PROFINIA™ proteinpurification protocol (Bio-Rad Laboratories, Inc.). The solublecomponents were loaded on a 5 ml His Trap column (Bio-Rad Laboratories,Inc.) preequilibrated with the same buffer used for bacterialresuspension; the soluble components were added at up to 5 ml/min(producing a “flow through fraction”) After loading on the column, thecolumn was washed with 10 column volumes of the same buffer at a rate of10 ml/min (producing a “wash fraction #1). A second wash using 20 mMbicine buffer or 20 mM HEPES buffer (pH 8.0) containing 500 mM NaCl and20 mM imidazole was performed, producing a “wash fraction #2). Elutionwas performed using 2 column volumes of 20 mM HEPES buffer or 50 mMbicine buffer (pH 8.0) containing 500 mM NaCl and 250 mM imidazole at arate of 10 ml/min, producing an “elution fraction”.

To improve the purity of the protein, positive elution fractions fromIMAC were pooled and loaded on a size exclusion chromatography (SEC)column (HILOAD™ SUPERDEX™ 200 26/60 from GE Healthcare) preequilibratedin phosphate buffered saline without calcium or magnesium (NaCl 137 mM,KCl 2.7 mM, Na₂HPO₄ 8.1 mM, KH₂PO₄ 1.47 mM, pH 7.4). Samples fromelution fractions were analyzed by sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE). Samples were concentrated usingCentricon 10 000 MW (Millipore).

Protein concentration was determined using spectrometer.

Example 5 SDS-PAGE and Western Blot Analysis of His Tapped Constructs &SDS-PAGE Analysis of Non-His Tapped LVL317 & LVL318 Constructs

Soluble and Insoluble Fraction Preparation

For example, 1 ml of culture after induction (see, for example, Example3 above) was centrifuged at 14 000 RPM for 2 min. The pellet wasresolubilized using 40 μl of BUGBUSTER® Protein Extraction Reagent(NOVAGEN®, EMD4 Biosciences, Merck), creating a cell suspension. Thecell suspension was incubated on a rotating platform for 10 min at roomtemperature. The cell suspension was then centrifuged at 14 000 RPM for2 min to separate the soluble fraction. The resulting pellet (insolublefraction) was resolubilized using 70 μl of deionized water, 5 μl ofdithiothreitol (DTT) 1M and 25 μl of NUPAGE® LDS (Lithium DodecylSulphate) Sample Buffer 4× (INVITROGEN™). The soluble fraction(supernatant from the cell suspension of the resolubilized pellet) wasadded to 30 μl of deionized water, 5 μl of DTT 1M and 25 μl of LDSSample Buffer 4×.

Media Fraction Preparation

For example, to prepare the media fraction, 100 μl of the supernatantfrom the induced whole cell culture following centrifugation (see, forexample, Example 3 above) was concentrated by adding 500 μl of RCreagent I (Bio-Rad Laboratories, Inc.); the sample was mixed andincubated for 1 min at room temperature. Then, 500 μl of Reagent II(Bio-Rad Laboratories, Inc.) was added to the sample and mixed. Thisformulation was centrifuged at 14 000 RPM for 10 min. The pellet wasresolubilized using 28 μl of deionized water, 2 μl of DTT 1M and 10 μlof LDS SB 4×.

Purification Fraction Preparation

For example, purified proteins (for example, obtained as described inExample 4) were prepared for SDS-PAGE analysis by adding 70 l of sample,5 l of DTT 1M and 25 l of LDS Sample Buffer 4×.

SDS-PAGE Analysis and Transfer to Nitrocellulose Membrane

SDS-PAGE analysis and transfer to nitrocellulose membrane were performedaccording to manufacturer's recommendations (Invitrogen) using NUPAGE®Bis-Tris 4-12% gels. Preparations of samples, buffers and migrationconditions were done under conditions recommended by the suppliers.

In one example, the gel was loaded with a 20 ul sample from a master mixcomprising 70 μl of a purified protein fraction, 5 μl of DTT 1M and 25μl of LDS SB 4×.

After samples were run on NUPAGE® Bis-Tris 4-12% gels, the proteins weretransferred to nitrocellulose membranes.

Nitrocellulose membranes were blocked for 30 minutes at 37° C., 60 RPMusing 3% milk/PBS 1× fresh solution. After the blocking incubation,Primary Antibodies were added (6×His Tag® antibody, Abcam PLC, cataloguenumber: ab9108) at a dilution of: 1:1000 in 3% milk/PBS 1× freshsolution for 1 hour at 37° C., 60 RPM. After that, membranes were washedthree times, for 5 minutes each, at room temperature using 0.02%polsorbate 20 (for example, TWEEN™ 20)/PBS 1×. Secondary Antibodies(alkaline phosphatase (AP) Rabbit anti-IgG (H+L) rabbit, JacksonImmunoResearch Laboratories, Inc.) were added at dilution 1:14 000 using3% milk/PBS 1× fresh solution. Membranes were incubated for 1 hour at37° C., 60 RPM. After that, membranes were washed three times for 5minutes at room temperature using 0.02% polysorbate 20 (for example,TWEEN™ 20)/PBS 1× before the membrane expositions to5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (forexample, BCIP®/NBT from Sigma-Aldrich®, 1 tablet/10 ml water).

See FIG. 1 for SDS-PAGE of induced bacterial extracts for fusion proteinconstructs LVL291, LVL268 and LVL269. Insoluble fraction (I), Solublefraction (S) and Culture Media fraction (M) were loaded for LVL291,LVL268 and LVL269 before and after induction (ind).

See FIG. 2 for SDS-PAGE and Western blot related to purificationextracts for fusion protein constructs LVL291, LVL268 and LVL269. Flowthrough fraction (Ft), Wash fraction (W) and Elution fraction (E) wereloaded for purification of LVL291, LVL268 and LVL269. Anti-his tag wasused to probe extracts.

See FIG. 3 for SDS-PAGE of induced bacterial and purification extractsfor fusion protein constructs LVL291 and LVL315. Culture Media fraction(M), Soluble fraction (Sol), Insoluble fraction (Ins), Flow throughfraction (Ft), Wash fraction #1 (W1), Wash fraction #2 (W2) and Elutionfraction (E) were loaded for LVL291 and LVL315.

See FIG. 4 for SDS-PAGE of induced bacterial and purification extractsfor fusion protein construct LVL312. Culture Media fraction (M), Solublefraction (Sol), Insoluble fraction (Ins), Flow Through fraction (Ft),Wash fraction #1 (W1), Wash fraction #2 (W2) and Elution fraction (E)were loaded for LVL312.

See FIG. 25 for SDS-PAGE of soluble fractions from induced bacterialextracts for fusion protein constructs LVL291, LVL702, LVL736, LVL737,LVL738, LVL739, LVL740 and pET26b vector (negative control). (a)Experiment 1 (b) Experiment 2 (c) Experiment 3. PE-PilA fusion proteinindicated by arrow.

See FIG. 26 for the average band percentage of fusion protein in thesoluble fraction from Experiments 1, 2 and 3.

LVL317 and LVL318 bacterial extracts used in the SDS-PAGE analysis inFIG. 5 and FIG. 6, respectively, were prepared generally as describedabove.

FIG. 5. SDS-PAGE of induced (1 mM and 10 μM IPTG) bacterial extracts forfusion protein construct LVL317. Extracts from before (NI) and afterinduction (In), Soluble fraction (S), Insoluble fraction (I).

FIG. 6. SDS-PAGE of induced (1 mM and 10 μM IPTG) bacterial extracts forfusion protein construct LVL318. Extracts from before (NI) and afterinduction (In), Culture Media fraction (M), Soluble fraction (S),Insoluble fraction (I).

Proteins separate by SDS-PAGE were transferred to an Immobilon-Pmembrane. The Coomassie Blue stained protein bands were cut and placedin a sequenator reactor. Sequencing was carried out according tomanufacturer's protocol using an Applied Biosystems PROCISE® ProteinSequencer, model 494-cLC.

TABLE 5 Shake flask protein expression profiles and signal peptidecleavage for fusion protein constructs. Fusion Protein Protein SignalConstruct Description Expression peptide ID N-term → C-term profilecleavage LVL312 FlgI sp - E - PilA fragment - GG - PE fragment - In: +++Confirmed GGHHHHHH So: + Se: + LVL291 PelB sp - PE fragment - GG - PilAfragment - In: +++ Confirmed GGHHHHHH So: ++ Se: + LVL268 PelB sp - D -PE fragment - GG - PilA fragment - In: +++ Confirmed GGHHHHHH So: ++Se: + LVL269 NadA sp - ATNDDD - PE fragment - GG - PilA In: +++Confirmed fragment - GGHHHHHH So: ++ Se: + LVL270 MHHHHHH - PEfragment - GG - PilA fragment In: + Not tested So: − Se: − LVL315 PelBsp - MD - PE fragment - GG - PilA fragment - In: +++ Confirmed GGHHHHHHSo: ++ Se: + LVL317 PelB - PE fragment - GG - PilA fragment In: +++Confirmed So: + Se: Nt LVL318 PelB sp - MD - PE fragment - GG - PilAfragment In: +++ So: + Se: − LVL702 PelB sp - PE fragment - GG - PilAfragment - In: +++ Confirmed GGHHHHHH So: ++ Se: Nt LVL736 PelB sp - PEfragment - GG - PilA fragment - In: +++ Confirmed GGHHHHHH So: ++ Se: NtLVL737 PelB sp - PE fragment - GG - PilA fragment - In: +++ ConfirmedGGHHHHHH So: ++ Se: Nt LVL738 PelB sp - PE fragment - GG - PilAfragment - In: +++ Confirmed GGHHHHHH So: ++ Se: Nt LVL739 PelB sp - PEfragment - GG - PilA fragment - In: +++ Confirmed GGHHHHHH So: ++ Se: NtLVL740 PelB sp - PE fragment - GG - PilA fragment - In: +++ ConfirmedGGHHHHHH So: ++ Se: Nt So = Soluble fraction. In = Insoluble fraction.Se = Protein Secreted in the media fraction. Nt = Not tested. Thefollowing rating were based on a visual inspection (coomassie) +: lowexpression; ++: medium expression; +++: high expression; −: noexpression

Example 6 LVL291 Fusion Protein Characterization

Physical Properties of LVL291: Folding of PE and PilA in LVL291 &Melting Point

Circular Dichroism:

Analysis of Secondary Structure

Circular dichroism (CD) is used to determine the secondary structurecomposition of a protein by measuring the difference in the absorptionof left-handed polarized light versus right-handed polarized light whichis due to structural asymmetry. The shape and the magnitude of the CDspectra in the far-UV region (190-250 nm) are different whether aprotein exhibits a beta-sheet, alpha-helix or random coil structure. Therelative abundance of each secondary structure type in a given proteinsample can be calculated by comparison to reference spectra.

Far UV spectra are measured using an optical path of 0.01 cm from 178 to250 nm, with a 1 nm resolution and bandwidth on a Jasco J-720spectropolarimeter. Temperature of the cell is maintained at 23° C. by aPeltier thermostated RTE-111 cell block. A nitrogen flow of 10 L/min ismaintained during the measurements.

Results:

The far-UV CD spectra obtained for PE (from construct pRIT16762), PilA(from construct pRIT 16790) and PE-PilA proteins are characteristic offolded proteins containing a mix of alpha and beta structures, but PE issignificantly richer in alpha helix than PilA and PE-PilA (FIG. 7, CDspectra of PE, PilA and PE-PilA fusion proteins).

In order to evaluate the integrity of the folding of PE and PilAindividual proteins once bound together in a chimeric protein and thenverify a possible interaction between both, difference spectra werecalculated.

-   -   When the PE and PilA far-UV spectra are combined, the resulting        spectrum superposes to the spectrum of PE-PilA chimer (FIG. 8,        Combination of PE and PilA CD spectrum). This result suggests        that the PE-PilA chimer contains all the secondary structures        that are detected in the individual components. It also suggests        that the fusion of the proteins has no major impact on the        secondary structures of the individual components and        consequently that the folding of PE and PilA is not        significantly different whether the proteins are separate or in        fusion.        Melting Point Evaluation:

In order to evaluate if the expression in fusion has an impact on thethermodynamic properties of the individual proteins, the melting pointsof PE, PilA and PE-PilA have been evaluated by monitoring the defoldingof the alpha helix with temperature by circular dichroism.

The presence of alpha helix is characterized by a minimum in theCircular dichroism signal at 222 nm, so a significant increase in CDsignal at 222 nm during temperature increase is an indication of proteindenaturation. The determination of the temperature at which the proteinundergoes loss in secondary structure allows the determination of themelting point (Tm), which corresponds to the temperature at which halfof the proteins have lost their structure.

Melting point can be determined by identification of the inflexion pointon the thermal denaturation curve obtained from a temperature versus CD222 nm plot.

-   -   Melting point of PilA and PE as determined by far-UV CD are        respectively of 52° C. and 68° C. (FIG. 9, PilA thermal        denaturation curve; FIG. 10, PE thermal denaturation curve).    -   The PE-PilA fusion protein exhibits two distinct Tm's at 48° C.        and 71° C. (FIG. 11, PE-PilA fusion protein thermal denaturation        curve). Those values indicate that the PE and PilA proteins are        still independently folded when bound into a chimer and that        they defold at a similar temperature whether they are separate        or in fusion. The observation that the defolding of the PilA        portion at 48° C. doesn't cause precipitation or impact the Tm        of the PE portion at 71° C. is a strong indication that the        interaction between PE and PilA within the fusion is minimal and        that they don't have a major observable impact on each other.        The melting points of proteins are sensitive to various external        conditions, including buffer composition or presence of        interacting molecules; that no major variation is observed upon        fusion of PE and PilA is a strong indication of the preservation        of most of the structure and of the properties of both PE and        PilA when they are bound together.

Example 7 Fermentation Process

Fusion proteins of the invention may be prepared by methods known bythose skilled in the art.

Example 8 Protein Purification of PE, PilA, and LVL317

PE Protein Purification from pRIT16762:

To generate the pRIT16762 expression vector, the pRIT16711 vector wasdigested using BamHI and NcoI restriction enzymes in order to delete 6amino acid residues between the signal sequence (pelB) and PE. Thevector obtained was named pRIT16712. In this vector, there are 3 aminoacids between the signal sequence pelB and PE: MDP. In a second step, asite directed mutagenesis was performed to change amino acid sequencefrom MDP to QIQ using pRIT16712 as template with primers MnoNTHi-44 andMnoNTHi-45 (described in Table 4) and the QuikChange II Site-DirectedMutagenesis Kit (Agilent Technologies, Stratagene Division).

Working seed of E. coli BLR(DE3) containing PE QIQ (from the pRIT16762construct) was thawed from −80° C. and used to prepare 100 ml ofpre-culture in LB broth by overnight incubation at 37° C. underagitation at 215 RPM. After overnight incubation, eight flaskscontaining 800 ml of LB APS were inoculated with 12.5 ml of pre-cultureand OD₆₀₀ measured at around 0.06. The cultures were incubated 3 h at37° C. with shaking. At a OD₆₀₀ of around 0.9, 1 mM IPTG was added tostart the induction. During the induction, the cultures were incubated19 h at 22° C. with shaking. After induction, OD₆₀₀ was at around 2.2.The cell cultures were transferred into 1 L centrifuge bags placedinside 1 L bottles and centrifuged at 4° C. for 30 minutes at 6,000×gand supernatant discarded. 1 ml aliquots of culture pre- andpost-induction and supernatant were kept for future analysis.

Lysis of the BLR(DE3) Induced with PE QIQ

The centrifuge bags were removed from the centrifugation bottles, openedand the pellet was expulsed from the bag into a beaker. The eightpellets were pulled together and resuspended in 100 ml of binding buffer(20 mM Hepes, 10 mM imidazole, 500 mM NaCl, pH 8.01). The E. coli BLR(DE3) containing the PE QIQ contruct were disrupted with the TS SeriesBench Top cell disrupter from Constant Systems Ltd. (1×30 kPsi; 1×15kPsi). The lysate was centrifuged 30 minutes, 6000 RPM, 4° C. Thesupernatant was kept and loaded on an IMAC column.

IMAC Purification of PE QIQ

IMAC column (BioRad, Bio-Scale Mini Profinity IMAC cartridge 5 ml) wasequilibrated with 5CV of Binding buffer (20 mM HEPES, 10 mM imidazole,500 mM NaCl, pH 8.01) at 5 ml/min. 100 ml of lysate supernatant wasloaded on the IMAC at 2.5 mL/min. Flow-through was collected in 50 mlfractions for future analysis. The column was washed with 3CV of Bindingbuffer to remove unbound protein. Sample containing unbound proteins wascollected in one aliquot of 15 ml in a 50 ml tube. The column was washedwith 2CV of Wash buffer (20 mM HEPES, 20 mM imidazole, 500 mM NaCl, pH8.01) collected in 2 ml fractions in a 96 well plate. The bound proteinwas then eluted with 6CV of 100% Elution buffer (20 mM HEPES, 250 mMimidazole, 500 mM NaCl, pH 8.01). The eluted protein was collected in 2ml fractions in 96-well plates. Wash and elution were performed at 5ml/min.

Size Exclusion Chromatography (SEC) on the IMAC Pool of PE QIQ

SEC column (GE healthcare, HILOAD™ 26/60 SUPERDEX™ 75 prep grade, 60 cmheight approx 319 ml volume) was equilibrated with 3CV of SEC buffer (20mM HEPES, 150 mM NaCl, pH8.49). 11 ml of IMAC eluate was loaded onto thecolumn at a flow rate of 2.5 ml/min. 2 ml fractions were collected from0.3CV to 0.9CV. Two runs were performed then fractions were analyzed bySDS-PAGE. Fractions from the two runs containing Prot E protein werepooled together (“SEC pool”, 48 ml approx total volume). 500 mM ofArginine was added to the SEC pool.

Dosage of the PE QIQ Pooled Samples Generated in the Above SEC Protocol

The SEC pool was dosed with the RCDC (Reducing Agent and DetergentCompatible) method from the Bio-Rad RC DC™ kit following manufacturer'sprotocol:

For each tested sample and standard, 25 μL was distributed in microfugetubes in duplicate. 125 μL of Bio-Rad RC Reagent I was added into eachtube; each tube was vortexed and incubate for 1 minute at roomtemperature. 125 μL of Bio-Rad RC Reagent II is added into each tube;each tube is vortexed and then centrifuged at 14,000×g for 5 minutes.Supernatants are discarded by inverting the tubes on clean, adsorbenttissue paper allowing the liquid to drain completely from the tubes.25.4 μL of Reagent A (already prepared by mixing 20 μL of Reagent S per1 ml of Reagent A) is added to each tube; each tube is vortexed andincubated at room temperature for 5 minutes, or until precipitate iscompletely dissolved. Vortex before proceeding to next step. Add 200 μLof DC reagent B to each tube and vortex immediately. Incubate at roomtemperature for 15 minutes. Transfer all samples to a 96-well plate andread the absorbance at 750 nm to determine the protein concentration foreach unknown protein sample.

The ProtE concentration was 1.069 mg/ml

PilA His-Tagged Protein Purification:

PilA was purified following the general procedure below:

-   -   E. coli cells containing a construct encoding PilA or a fragment        thereof are suspended in BUGBUSTER® and BENZONASE® nuclease        (NOVAGEN®), for example 10 ml BUGBUSTER® and 10 ul BENZONASE®        nuclease. The cell lysate is mixed at room temperature on a        rotating platform, for example, for 15 minutes. The cell lysate        is centrifuged at 4° C., for example at 16,000 g for 20 minutes.        The supernatant containing the protein is added to a Ni NTA        column containing Ni NTA HIS.BIND® resin and mixed at 4° C., for        example for 1 hour. The column may consist of 2 ml of Ni NTA        HIS.BIND® resin (NOVAGEN®) and 10 ml 1× Binding Buffer (from        NOVAGEN®'s Ni-NTA Buffer Kit). The column flow through is then        collected. The resin is washed two times with 1× wash buffer,        for example, containing 300 mM NaCl, 50 mM NaH₂PO₄, 25 mM        imidazone, pH 8.0). The wash is collected by gravity flow. The        protein is eluted from the column with 1× elution buffer, for        example, 300 mM NaCl, 50 mM NaH₂PO₄, 250 mM imidazone, pH 8.0.        The protein may be further purified by dialysis with the Binding        Buffer and rerun over a Ni NTA column as described above.        Thrombin Cleavage of PilA.

PilA is then incubated with thrombin (diluted 1/50) at room temperaturefor 16 h, to remove the histidine tag.

Size Exclusion Chromatography (SEC) on PilA Cleaved with Thrombin.

SEC column (GE healthcare, HILOAD™ 26/60 SUPERDEX™ 75 prep grade, 60 cmheight approx 319 ml volume) was equilibrated with 5CV of SEC buffer (20mM HEPES, 150 mM NaCl, pH8.52). Approximately 10 ml of cleaved PilA wasloaded onto the column at a flow rate of 2.5 ml/min. 2 ml fractionscollected from 0.3CV to 0.9CV. Two runs were performed then fractionswere analyzed by SDS-PAGE. Fractions from the two runs containingcleaved PilA protein were pooled together (“SEC pool”, 52 ml approxtotal volume).

Dosage of PilA, SEC Pool.

The SEC pool was dosed with the RCDC method as described above. Thecleaved PilA concentration was at 5.37 mg/ml.

Dialysis of the PilA SEC Pool with PBS 1× pH 7.4 (Dialysis Factor=1600)and Dosage by RCDC

The concentration post-dialysis determined by RCDC was at 3.0 mg/ml.

Purification of LVL317

Osmotic Shock

Since LVL317 fusion protein is expressed and processed in bacterialperiplasm, the protein was extracted by osmotic shock.

Frozen (−20° C.) harvested E. coli B2448 cell paste containing LVL317from 4 L of fermentor culture were pooled and resuspended in ahypertonic buffer consisting of 24 mM Tris-HCl, 16% (w/v) sucrose, 9.9%(w/v) glucose, 10 mM EDTA, pH 8.0 up to a final volume of 4 L. Thesuspension was mixed gently for 30 min at room temperature using a3-blade propeller installed on RW 16 basic stirrer, at medium speed. Thesuspension was centrifuged at 15,900×g for 30 minutes at roomtemperature. Supernatant (SN1) was kept for gel analysis.

The resulting pellet was resuspended in a hypotonic solution; 38 mMMgCl₂, and mixed for 30 min at room temperature. The mixture wascentrifuged at 15,900×g for 30 minutes at room temperature and theantigen recovered in the supernatant (SN2).

A clarification of the SN2 was performed by filtration through a0.45/0.2 μm polyethersulfone Sartorius Sartopore 2 MidiCap filter, at600 ml/min of flow rate.

The SN2 was diluted 1:3 with 20 mM NaH₂PO₄—Na₂HPO₄, pH 7.0, the pHadjusted to 7.0 if necessary and another clarification by filtrationthrough a 0.45/0.2 μm polyethersulfone Sartorius Sartopore 2 MidiCapfilter, at 600 ml/min was performed.

SP SEPHAROSE™ Fast Flow (SP FF) Chromatography

The diluted/filtered SN2 was loaded and captured on a strong cationicexchanger resin (SP SEPHAROSE™ FF-GE Healthcare) in a 14 cm ID (internaldiameter)×20 cm length column (column volume 3100 ml) equilibrated with2CV of 20 mM NaH₂PO₄/Na₂HPO₄ buffer pH 7.0. After washing the columnwith 5CV of 20 mM NaH₂PO₄/Na₂HPO₄ buffer pH 7.0, the antigen (containedwithin LVL317) was eluted by increasing the concentration of NaCl up to100 mM in the same washing buffer.

See FIG. 12 for a typical SP SEPHAROSE™ Fast Flow chromatogram.

Q SEPHAROSE™ Fast Flow (Q FF) Chromatography

The antigen present in the SP FF Eluate was diluted 1:4 with a 20 mMTris pH 8.5, pH adjusted to 8.5 if necessary and passed through a stronganionic exchanger resin (Q SEPHAROSE™ FF-GE Healthcare) in a 14 cmID×11.8 cm length column (column volume 1800 ml) equilibrated with 2CVof 20 mM Tris buffer pH 8.5. The antigen was recovered in theflow-through fraction.

See FIG. 13 for a typical Q SEPHAROSE™ Fast Flow chromatogram.

Concentration, Diaflitration, Polysorbate 80 Addition and SterileFiltration

The Q FF flow-through containing the antigen was concentrated up to0.7-0.8 mg/ml based on chromatogram UV and diafiltered with 5DV of 10 mMKH₂PO₄/K₂HPO₄ buffer pH 6.5 using a Pellicon-2™ 10 kDa cutoff membrane(Millipore).

Using a 5% stock solution, polysorbate 80 (for example, TWEEN™ 80) wasadded to the ultrafiltration retentate and agitated for 30 minutes withmagnetic stirrer at 130 rpm at 4° C. The final concentration ofpolysorbate 80 was 0.04%. Ultrafiltration retentate was sterilized byfiltration through a 0.45/0.2 μm Cellulose Acetate membrane (Sartobran300, Sartorius). The purified bulk was stored at −20° C. or −80° C.Absolute protein concentration was measured by AAA (Amino Acid Analysis)at 0.737 mg/ml.

Example 9 Use of Polysorbate 80

A titration experiment indicated that the addition of polysorbate 80,specifically, TWEEN™ 80 to a final concentration of 0.04% (w/v) to thepurified bulk prior to sterile filtration reduced filamentous particleformation and aggregation.

According to DSC analysis, TWEEN™ 80 reduced the degree of structuralchange (30-45° C.) seen after freeze/thaw cycles after storage at −20°C. and after storage 4 days at 4° C., −20° C. and −80° C. and 37° C.

Example 10 SDS-PAGE and Western Blot Analysis of LVL317

SDS-PAGE and Western Blot Analysis:

NUPAGE®, Bis-Tris 4-12% gel was loaded as described below with 10 μg ofsample in NUPAGE® LDS sample buffer containing 50 mM DTT heated 5 min at95° C. (20 μL of sample was loaded for samples having lowconcentration). Migration: 35 minutes at 200 Volts at room temperature(RT) in NUPAGE® MES Running Buffer. Gel Stained 2 hours in Instant blue(Novexin cat.: ISB01L) and destained overnight in water.

Lane Contents:

1: MW standard (10 μL) 2: Start (total fraction) (10 μg) 3: SN1 nonfiltered (10 μg) 4: SN2 not filtered (10 μg) 5: Not extracted (10 μg) 6:Load SP FF (10 μg) 7: Flow through SP FF (6.9 μg) 8: Wash SP FF (20 μL)9: Elution SP FF (10 μg) 10: Strip SP FF (10 μg) 11: Load Q FF (8.9 μg)12: Elution Q FF (9.8 μg) 13: Strip Q FF (4.8 μg) 14: TFF retentatebefore0.04% TWEEN ™ 80 spiked (10 μg) 15: Purified bulk Not filtered0.04% TWEEN ™ 80 spiked (10 μg) 16: Purified bulk Sterile Filtered 0.04%TWEEN ™ 80 spiked (10 μg) 17: Purified bulk Sterile Filtered 0.04%TWEEN ™ 80 spiked (20 μg + spiked E. Coli Cell lysate Rix (1 μg)) 18: E.Coli Cell lysate Rix (2 μg) 19: E. Coli Cell lysate Rix (1 μg) 20: E.Coli Cell lysate Rix (0.5 μg)

See FIG. 14 for a SDS-PAGE of In-process samples from purificationprocess of PE-PilA fusion protein.

For Western Blot, proteins were transferred at 4° C. overnight at 30Volts in NUPAGE® transfer buffer+20% Methanol, 0.1% SDS onnitrocellulose membrane. Membranes were blocked 1 hour with 50 mM Tris,150 mM NaCl pH 7.4+5% non-fat dry milk, incubated 2 hours in rabbitpolyclonal primary antibody diluted in blocking buffer (anti-Prot-E 1/50000 and anti-E coli (BLR) 1/1 000), washed 3×5 minutes in 50 mM Tris pH7.4+0.05% Tween 20, incubated 1 hour in secondary antibody (goatanti-rabbit conjugated to alkaline phosphatase diluted 1/5000 inblocking buffer), washed 3×5 minutes in wash buffer and developed inBCIP/NBT substrate (1 tablet per 10 ml). All incubations performed in 25ml per membrane.

See FIG. 15 for a Western Blot of In-process samples of purificationprocess from PE-PilA fusion protein. Blot using rabbit polyclonalanti-PE.

Lane Contents:

1: MW standard (10 μL) 2: Start (total fraction) (10 μg) 3: SN1 nonfiltered (10 μg) 4: SN2 not filtered (10 μg) 5: Not extracted (10 μg) 6:Load SP FF (10 μg) 7: Flow through SP FF (6.9 μg) 8: Wash SP FF (20 μL)9: Elution SP FF (10 μg) 10: Strip SP FF (10 μg) 11: Load Q FF (8.9 μg)12: Elution Q FF (9.8 μg) 13: Strip Q FF (4.8 μg) 14: TFF retentatebefore0.04% TWEEN ™ 80 spiked (10 μg) 15: Purified bulk Not filtered0.04% TWEEN ™ 80 spiked (10 μg) 16: Purified bulk Sterile Filtered 0.04%TWEEN ™ 80 spiked (10 μg) 17: Purified bulk Sterile Filtered 0.04%TWEEN ™ 80 spiked (20 μg + spiked E. Coli Cell lysate Rix (1 μg)) 18: E.Coli Cell lysate Rix (2 μg) 19: E. Coli Cell lysate Rix (1 μg) 20: E.Coli Cell lysate Rix (0.5 μg)

See FIG. 16 for a Western Blot of In-process samples of purificationprocess from PE-PilA fusion protein. Blot using rabbit polyclonalanti-E. coli (BLR).

Lane Contents:

1: MW standard (10 μL) 2: Start (total fraction) (10 μg) 3: SN1 nonfiltered (10 μg) 4: SN2 not filtered (10 μg) 5: Not extracted (10 μg) 6:Load SP FF (10 μg) 7: Flow through SP FF (6.9 μg) 8: Wash SP FF (20 μL)9: Elution SP FF (10 μg) 10: Strip SP FF (10 μg) 11: Load Q FF (8.9 μg)12: Elution Q FF (9.8 μg) 13: Strip Q FF (4.8 μg) 14: TFF retentatebefore0.04% TWEEN™ 80 spiked (10 μg) 15: Purified bulk Not filtered0.04% TWEEN ™ 80 spiked (10 μg) 16: Purified bulk Sterile Filtered 0.04%TWEEN ™ 80 spiked (10 μg) 17: Purified bulk Sterile Filtered 0.04%TWEEN ™ 80 spiked (20 μg + spiked E. Coli Cell lysate Rix (1 μg)) 18: E.Coli Cell lysate Rix (2 μg) 19: E. Coli Cell lysate Rix (1 μg) 20: E.Coli Cell lysate Rix (0.5 μg)

SDS-PAGE and Western Blot Figures Comments:

The PE-PilA fusion protein migrates at 30 kDa. The extraction by osmoticshock extracts the fusion protein expressed and processed in bacteriaperiplasm and reduced contamination from bacteria. Small loss of fusionprotein during hypertonic treatment (lane 3). A small proportion is notextracted by hypotonic treatment and remains associated with cells (lane5). Small loss in SP FF Flow through (lane 7) and in strip fraction ofboth columns (lanes 10 and 13). Since the total volume of strip fractionis low the loss of fusion protein is not significant. Degraded bands arevisible in strip fractions but not in final product. No significantcontamination from E. coli host cell proteins in purified bulk (lane16).

Analysis of LVL735 and LVL778 yielded similar profiles as LVL317.

Example 11 Melting Point Data for PE, PilA and LVL317

Thermal transition of PE-PilA fusion non His-tagged protein (LVL317) wascompared with the thermal transition of both PE his-tagged (as describedin Example 8) and cleaved PilA (as described in Example 8) proteins,purified as described above.

Before DSC, PE and PilA were dialyzed overnight in 10 mM K₂HPO₄/KH₂PO₄pH 6.5+0.04% Tween 80 (1:250 sample:buffer volume ratio) to have them inthe same buffer as the fusion protein. After dialysis, proteinsconcentration was measured by BCA and adjusted to 300 μg/ml (PE) and 500μg/ml (PilA).

Analysis done on VP™-DSC from MicroCal, LLC (part of GE Healthcare). Thefinal dialysis buffer was used as reference and subtracted from thescans. DSC scan rate 90° C./hr. In order to evaluate the capacity tomeasure the thermal transition in the Final Container (FC) afterformulation, the fusion protein was diluted to the FC concentration (60μg/ml). Final container data not shown.

Results:

See FIG. 17 for Thermal transition of PE-PilA fusion protein and PE andPilA proteins. Curves: PilA (1), Protein E (Prot E, PE) (2), PE-PilA PBnot diluted 737 μg/ml (3), and PE-PilA PB diluted at FC concentration 60μg/ml (4).

1—PilA Tm: 53° C. 2—Protein E Tm: 63 3—PE-PilA PB (Purified Bulk) Tm₁:53.7° C. and Tm₂: 66.1° C. not diluted 737 μg/ml 4—PE-PilA PB diluted atFC Tm1: 53.2° C. and Tm2: 67.6° C. concentration 60 μg/ml

Two transitions were detected in the purified fusion protein (LVL317)(curves 3 and 4).

The Tm₁ (53.7° C.) of the PE-PilA fusion protein is similar to PilAtransition (53° C.).

Significant shift of Tm₂ in PE-PilA (66.1° C.) as compared to PEtransition (63° C.). The fusion of both domains seems to stabilize thePE fragment.

The shift of Tm₂ in the diluted fusion protein as compared to undilutedis a concentration artifact arising from the steep decreasing slopetypical of aggregation which is concentration dependant.

Antigen folding analysis of LVL735 and LVL778 were similar to that ofLVL317.

Example 12 PE-PilA Fusion Protein Construct LVL291 Anti-PilAImmunogenicity Response in Balb/c Mice

The immune response directed against purified LVL291 PE-PilA fusionprotein (the LVL291 fusion protein without the heterologous signalpeptide) formulated in AS03_(A) was evaluated in Balb/c mice. Animals(20 mice/group) were immunized by the intramuscular route at days 0, 14and 28 with 10 μg of PE (from vector pRIT16762), PilA (from vectorpRIT16790) or PE-PilA, each formulated in AS03_(A). The control groupwas vaccinated with AS03_(A) alone. Antibody response directed againsteach antigen was determined in individual sera collected at day 42. Noantibody response was obtained with the negative control. As shown inFIG. 18, the antibody response directed against PilA was higher in miceimmunized with the PE-PilA fusion compared to antibody response in miceimmunized with monovalent PilA. The antibody responses directed againstPE were similar in mice immunized with the fusion protein and miceimmunized with monovalent PE. GMT=geometric means titer. Data werecaptured and analyzed with the SOFTMAX® Pro Software (Molecular Devices)running under WINDOWS®(Microsoft); the four parameters logistic logfunction was used to calculate the standard curve. The four-parameterlogistic-log function describes, with a high degree of accuracy, thecurve of the reference serum displaying a pronounced sigmoïdal shapewhen plotted on an optical density-versus-concentration (log) scale.Antibody concentrations were calculated at each dilution of mice serumsamples by interpolation of the standard curve. The antibody in qualitycontrol sera and in unknown serum samples is obtained by averaging thevalues from all dilutions that fall within the working range (10-80%) ofthe dilution curve of the reference. Results are shown in FIG. 18, whichgraphs the antibody responses against LVL291 PE-PilA fusion protein andagainst monovalent PE and PilA in the Balb/c mouse model.

Example 13 Murine Nasopharyngeal Colonization Model. Immunization withPE-PilA. Challenge with NTHi Strain 86-028NP and NTHi Strain 3224A

Balb/c female mice (20/group) were immunized intranasally at days 0 and14 with 6 μg of a purified PE-PilA fusion protein (LVL291 for challengewith 86-028NP; LVL317 for challenge with strain 3224A) formulated withLT (heat labile toxin of Escheria coli) and on day 28 with 6 μg of apurified PE-PilA fusion protein in phosphate buffered saline (PBS).Control mice (20/group) were vaccinated with LT alone. Mice weresubsequently challenged intranasally with 5×10⁶ CFU (colony formingunits) of homologous NTHi strain 86-028NP and heterologous NTHi strain3224A. Homology and heterology are determined by reference to the NTHistrain with which the mice were immunized. Bacterial colonies werecounted in nasal cavities removed 1 and 2 days after the challenge.D1=day 1. D2=day 2. PE-PilA vaccination increased the clearance of NTHistrain 86-028NP and strain 3224A in the nasopharynx at day 1 and day 2post challenge.

For the experiment performed with NTHi strain 86-028NP: A 2-way fixedANOVA was performed using the log 10 values of the counts as response,the fixed factors being the group (4 levels) and the day (2 levels). Theassumption of variance heterogeneity was rejected and a model withheterogeneous variances was fitted to the data. No significantinteraction was detected between the 2 factors. The group fusion PE-PilA(6 μg per mouse) significantly reduced CFU compared with the controlgroup (LT); the geometric mean ratio being equal to 0.06 with a 95%confidence interval of 0.01, 0.25.

For the experiment conducted with NTHi strain 3224A: A 3-way fixed ANOVAwas performed using the log 10 values as response, the fixed factorsbeing the group, the day, and the experiment. The Shapiro-Wilk andLevene's test did not reject the assumptions of normality and ofhomogeneity of variances. No significant interaction between any of the2 factors or between the 3 factors was detected and only main factorswere kept in the analysis. PE-PilA/LT significantly reduced CFU comparedwith the control group; the geometric mean ratio being equal to 0.11with a 95% confidence interval of 0.02, 0.61.

See FIG. 19 for effect of PE-PilA fusion protein vaccination on NTHistrain 86-028NP bacterial clearance in mouse nasopharynx.

See FIG. 20 for effect of PE-PilA fusion protein vaccination on NTHistrain 3224A bacterial clearance in mouse nasopharynx.

Example 14 Murine Nasopharyngeal Colonization Model. Immunization withPilA. Challenge with NTHi Strain 3219C

Female OF1 mice (20 mice/group) were immunized intranasally at days 0and 14 with 3 μg PilA (from vector 16790) formulated with LT and at day28 with 3 μg PilA in PBS. Control mice were vaccinated with LT alone.Mice were subsequently challenged intranasally with 5×10⁶ CFU of NTHistrain 3219C. Bacterial colonies were counted in nasal cavities removed3 and 4 days after the challenge. D3=day 3. D4=day 4.

See FIG. 21 for effect of PilA vaccination on bacterial clearance inmouse nasopharynx.

Example 15 Murine Nasopharyngeal Colonization Model. Immunization withPE. Challenge with NTHi Strain 3224A

Balb/c female mice (20 mice/group) were immunized intranasally at days 0and 14 with 3 μg PE (from vector pRIT16762) formulated with LT and atday 28 with 3 μg PE in PBS. Control mice were vaccinated with LT alone.Mice were subsequently challenged intranasally with 5×10⁶ CFU of NTHistrain 3224A. Bacterial colonies were counted in nasal cavities removed3 and 4 days after the challenge. 10 mice were examined on day 3 (D3).10 mice were examined on day 4 (D4). PE vaccination increasedsignificantly the clearance of NTHi in the naso-pharynx at day 4 postchallenge (FIG. 22), using on the Dunn test for statistical analysis.

See FIG. 22 for effect of PE vaccination on bacterial clearance in thenasopharynx of mice.

Example 16 Vibronectin Binding. Inhibition of Vibronectin Binding byLVL317 & LVL735 PE-PilA Fusion Protein

The ability of PE in the purified LVL317 PE-PilA fusion proteinconstruct to bind to vitronectin was evaluated. Microtiter plates(POLYSORP™, Nunc, Thermo Fisher Scientific) were coated with PE (fromvector pRIT16762) or with purified LVL317 PE-PilA fusion protein (10μg/ml). Plates were washed four times with NaCl 150 mM-polysorbate 20,0.05% (for example, TWEEN™ 20) and blocked for one to two hours withPBS-BSA 1%. After four washings, vitronectin (Vitronectin from humanplasma, SIGMA-ALDRICH®) was added (10 μg/ml), two fold diluted (12dilutions), and the plates were incubated for 1 h at room temperature.The plates were then washed 4 times with NaCl 150 mM-polysorbate 20,0.05% (for example TWEEN™ 20) After washings, the bound vitronectin wasdetected using peroxydase sheep anti-human vitronectin (US Biological)followed by the addition of ortho-phenylene diamine/H₂O₂ substrate. Thecolor developed is directly proportional to the amount of antibody fixedto the vitronectin.

See FIG. 23 for (a) LVL317 PE-PilA fusion protein bound to vitronectin.PilA=PilA from NTHi strain 86-028NP (as described for pRIT16790);PE=Protein E (as described for pRIT16762) and (b) LVL317 and LVL735PE-PilA fusion protein bound to vitronectin.

Example 17 Vibronectin Binding. Inhibition of Vibronectin Binding byAntibodies Directed Against the LVL291 PE-PilA Fusion Protein

Microtiter plates (POLYSORP™, Nunc, Thermo Fisher Scientific) werecoated with PE (from vector pRIT16762) or with purified PE-PilA fusionprotein (10 μg/ml). Plates were washed four times with NaCl 150mM-polysorbate 20, 0.05% (for example, TWEEN™ 20) and blocked for twohours with PBS-BSA 1%. After washings, vitronectin (Vitronectin fromhuman plasma, SIGMA-ALDRICH®) was added at 50 μg/ml and purifiedantibodies anti-PE-PilA (produced and purified in house) were two-foldserially diluted and incubated for 1 h at room temperature. The plateswere then washed 4 times with NaCl 150 mM-polysorbate 20, 0.05% (forexample, TWEEN™ 20). After four washings, the bound vitronectin wasdetected using peroxydase sheep anti-Vitronectin (US Biological)followed by the addition of ortho-phenylene diamine/H₂O₂ substrate. Thecolor developed is directly proportional to the amount of antibody fixedto the vitronectin.

Inhibition of vitronectin binding to PE by polyclonal antibodiesdirected against PE-PilA was observed.

See FIG. 24 for inhibition of vitronectin binding by polyclonalantibodies against PE-PilA fusion protein.

Example 18 Antigenicity of LVL291 PE-PilA Fusion Protein. ELISA

Purified LVL291 PE-PilA fusion protein was validated in an antigenicitytest with monovalent proteins as control. The fusion protein was testedin a sandwich ELISA developed with polyclonal antibodies (rabbit andguinea pig) generated against the PE gene fragment coding for aminoacids 22 to 160 of SEQ ID NO: 4 (as described for pRIT16711) or againstPilA from NTHi strain 86-028NP (from vector pRIT16790).

PilA or PE was added at 100 ng/ml and serially two fold diluted. After30 minutes incubation and after washing, the bound antigen was detectedby a rabbit polyclonal serum obtained after immunisation with PE orPilA. The bound antibodies were detected using a peroxydase anti-rabbitIg (Jackson ImmunoResearch Laboratories, Inc.) followed by the additionof ortho-phenylene-diamine/H₂O₂ substrate. The color developed isdirectly proportional to the amount of antigen present. Absorbancereadings were measured using a spectrophotometer for microtiter plates.The antigenicity of the samples was determined by comparison to thecurve of the full length PE or full length PilA reference antigen and isexpressed in ug/ml. The reference represented 100% of antigenicity.

As observed in the Table 6: Antigenicity was observed with the purifiedLVL291 PE-PilA fusion protein compared to the monovalent PE and PilAantigens.

TABLE 6 Relative antigenicity obtained with purified LVL291 PE-PilAfusion protein in the antigenicity test. PE relative antigenicity (%)Protein E as Reference 100 PE-PilA 130-148 PilA as Reference 100 PE-PilA120-152

Example 19 Immunogenicity of LVL735 PE-PilA Fusion Protein

Female Balb/c mice (n=34) were immunized by the intramuscular route atdays 0, 14 and 28 with 50 μl of vaccine formulation containing 1, 0.2 or0.04 μg of PE-PilA fusion protein LVL317 or LVL735 formulated withinAS01_(E) or AIPO₄ (aluminium phosphate). The antibody responses to PEand PilA were determined in individual sera collected at day 42 and theIgG level against PE and PilA was measured and expressed in μg/ml.

See FIG. 27 for PE and PilA antibody response to LVL317 and LVL735.GMC=geometric mean concentration. GMT=geometric means titer.IC=confidence intervals.

Example 20 Protective Efficacy of the LVL735 and LVL317 Fusion Proteinsin a Mouse Model of Non-Typeable Haemophilus influenzae NasopharyngealColonization

Female Balb/c mice were intranasally immunized at days 0 and 14 with 10μl of vaccine formulation containing 5.8 μg of LVL735 or LVL317 admixedwith 0.5 μg of E. coli labile toxin (LT). A booster dose of 5.8 μg ofnon-adjuvanted LVL735 or LVL317 was administered at day 28. Control micewere vaccinated with LT alone at days 0 and 14, and PBS at day 28.Animals were intranasally challenged with 5×10⁶ cfu of NTHi 3224A strainat day 42. Bacterial colonies were counted in nasal cavities removed 1and 2 days after the challenge (n=10/time-point).

Nasal cavities are homogenized in medium and a bacterial quantificationis performed. Results are well expressed in CFU/ml.

See FIG. 28 for the effect of LVL735 and LVL317 vaccination on bacterialclearance in a mouse model of non-typeable Haemophilus influenzaenasopharyngeal colonization.

Example 21 Formulation of Multivalent Vaccines Comprising a PE-PilAFusion Protein

3 vaccines were designed:

10V:

A ten valent (10V) vaccine containing the following ten S. pneumoniaecapsular saccharide conjugates: capsular saccharide from serotype 1conjugated to protein D (1-PD), capsular saccharide from serotype 4conjugated to protein D (4-PD), capsular saccharide from serotype 5conjugated to protein D (5-PD), capsular saccharide from serotype 6Bconjugated to protein D (6B-PD), capsular saccharide from serotype 7Fconjugated to protein D (7F-PD), capsular saccharide from serotype 9Vconjugated to protein D (9V-PD), capsular saccharide from serotype 14conjugated to protein D (14-PD), capsular saccharide from serotype 23Fconjugated to protein D (23F-PD), capsular saccharide from serotype 18Cconjugated to tetanus toxoid (18C-TT) and capsular saccharide fromserotype 19F conjugated to Diphtheria Toxin (19F-DT).

12V:

A twelve valent (12V) vaccine containing the same ten S. pneumoniaecapsular saccharide conjugates as 10V with an additional two S.pneumoniae saccharide conjugates, 19A conjugated to CRM197 (19ACRM) and6A conjugated to CRM197 (6ACRM).

12V+ Proteins (12V+prot):

A vaccine containing the same twelve S. pneumoniae capsular saccharideconjugates as 12V with the addition of PhtD, dPly and PE-PilA fusionprotein.

Preparation of dPly:

Pneumococcal pneumolysin was prepared and detoxified as described inWO2004/081515 and WO2006/32499 using formaldehyde detoxification.

Expression and Purification of PhtD:

Expression of PhtD:

The PhtD protein is a member of the pneumococcal histidine-triad (Pht)protein family characterized by the presence of histidine-triads. PhtDis a 838 aa-molecule and carries 5 histidine triads (see MedImmuneWO00/37105 SEQ ID NO: 4 for amino acid sequence and SEQ ID NO: 5 for DNAsequence). PhtD also contains a proline-rich region in the middle (aminoacid position 348-380). PhtD has a 20 aa-N-terminal signal sequence.Preparation and purification of PhtD is described in WO2007/071710 (see,for example, Example 1b). The sequence of amino acids 21-838 of SEQ IDNO:4 from WO00/37105 corresponds to SEQ ID NO:220.

SEQ ID NO: 220Ser Tyr Glu Leu Gly Arg His Gln Ala Gly Gln Val Lys Lys Glu Ser Asn Arg Val Ser Tyr IleAsp Gly Asp Gln Ala Gly Gln Lys Ala Glu Asn Leu Thr Pro Asp Glu Val Ser Lys Arg Glu GlyIle Asn Ala Glu Gln Ile Val Ile Lys Ile Thr Asp Gln Gly Tyr Val Thr Ser His Gly Asp His TyrHis Tyr Tyr Asn Gly Lys Val Pro Tyr Asp Ala Ile Ile Ser Glu Glu Leu Leu Met Lys Asp Pro AsnTyr Gln Leu Lys Asp Ser Asp Ile Val Asn Glu Ile Lys Gly Gly Tyr Val Ile Lys Val Asp Gly LysTyr Tyr Val Tyr Leu Lys Asp Ala Ala His Ala Asp Asn Ile Arg Thr Lys Glu Glu Ile Lys ArgGln Lys Gln Glu His Ser His Asn His Gly Gly Gly Ser Asn Asp Gln Ala Val Val Ala Ala ArgAla Gln Gly Arg Tyr Thr Thr Asp Asp Gly Tyr Ile Phe Asn Ala Ser Asp Ile Ile Glu Asp Thr GlyAsp Ala Tyr Ile Val Pro His Gly Asp His Tyr His Tyr Ile Pro Lys Asn Glu Leu Ser Ala Ser GluLeu Ala Ala Ala Glu Ala Tyr Trp Asn Gly Lys Gln Gly Ser Arg Pro Ser Ser Ser Ser Ser Tyr AsnAla Asn Pro Ala Gln Pro Arg Leu Ser Glu Asn His Asn Leu Thr Val Thr Pro Thr Tyr His GlnAsn Gln Gly Glu Asn Ile Ser Ser Leu Leu Arg Glu Leu Tyr Ala Lys Pro Leu Ser Glu Arg HisVal Glu Ser Asp Gly Leu Ile Phe Asp Pro Ala Gln Ile Thr Ser Arg Thr Ala Arg Gly Val Ala ValPro His Gly Asn His Tyr His Phe Ile Pro Tyr Glu Gln Met Ser Glu Leu Glu Lys Arg Ile Ala ArgIle Ile Pro Leu Arg Tyr Arg Ser Asn His Trp Val Pro Asp Ser Arg Pro Glu Gln Pro Ser Pro GlnSer Thr Pro Glu Pro Ser Pro Ser Pro Gln Pro Ala Pro Asn Pro Gln Pro Ala Pro Ser Asn Pro IleAsp Glu Lys Leu Val Lys Glu Ala Val Arg Lys Val Gly Asp Gly Tyr Val Phe Glu Glu Asn GlyVal Ser Arg Tyr Ile Pro Ala Lys Asp Leu Ser Ala Glu Thr Ala Ala Gly Ile Asp Ser Lys Leu AlaLys Gln Glu Ser Leu Ser His Lys Leu Gly Ala Lys Lys Thr Asp Leu Pro Ser Ser Asp Arg GluPhe Tyr Asn Lys Ala Tyr Asp Leu Leu Ala Arg Ile His Gln Asp Leu Leu Asp Asn Lys Gly ArgGln Val Asp Phe Glu Ala Leu Asp Asn Leu Leu Glu Arg Leu Lys Asp Val Pro Ser Asp Lys ValLys Leu Val Asp Asp Ile Leu Ala Phe Leu Ala Pro Ile Arg His Pro Glu Arg Leu Gly Lys ProAsn Ala Gln Ile Thr Tyr Thr Asp Asp Glu Ile Gln Val Ala Lys Leu Ala Gly Lys Tyr Thr Thr GluAsp Gly Tyr Ile Phe Asp Pro Arg Asp Ile Thr Ser Asp Glu Gly Asp Ala Tyr Val Thr Pro His MetThr His Ser His Trp Ile Lys Lys Asp Ser Leu Ser Glu Ala Glu Arg Ala Ala Ala Gln Ala Tyr AlaLys Glu Lys Gly Leu Thr Pro Pro Ser Thr Asp His Gln Asp Ser Gly Asn Thr Glu Ala Lys GlyAla Glu Ala Ile Tyr Asn Arg Val Lys Ala Ala Lys Lys Val Pro Leu Asp Arg Met Pro Tyr AsnLeu Gln Tyr Thr Val Glu Val Lys Asn Gly Ser Leu Ile Ile Pro His Tyr Asp His Tyr His Asn IleLys Phe Glu Trp Phe Asp Glu Gly Leu Tyr Glu Ala Pro Lys Gly Tyr Thr Leu Glu Asp Leu LeuAla Thr Val Lys Tyr Tyr Val Glu His Pro Asn Glu Arg Pro His Ser Asp Asn Gly Phe Gly AsnAla Ser Asp His Val Arg Lys Asn Lys Val Asp Gln Asp Ser Lys Pro Asp Glu Asp Lys Glu HisAsp Glu Val Ser Glu Pro Thr His Pro Glu Ser Asp Glu Lys Glu Asn His Ala Gly Leu Asn ProSer Ala Asp Asn Leu Tyr Lys Pro Ser Thr Asp Thr Glu Glu Thr Glu Glu Glu Ala Glu Asp ThrThr Asp Glu Ala Glu Ile Pro Gln Val Glu Asn Ser Val Ile Asn Ala Lys Ile Ala Asp Ala Glu AlaLeu Leu Glu Lys Val Thr Asp Pro Ser Ile Arg Gln Asn Ala Met Glu Thr Leu Thr Gly Leu LysSer Ser Leu Leu Leu Gly Thr Lys Asp Asn Asn Thr Ile Ser Ala Glu Val Asp Ser Leu Leu AlaLeu Leu Lys Glu Ser Gln Pro Ala Pro IleExpression of Protein D

Protein D was expressed as described in WO2007/071710

Expression and Purification of CRM197 E. coli:

In order to improve the yield of process for the production of CRM197,alternative expression modes were evaluated with a target of a 10-foldincrease of process yield. The selected construction was expressed inEscherichia coli strain (B834 (DE3)) as a fusion between FlgI signalsequence from E. coli (19aa) and CRM197 (537aa). The signal sequence iscleaved upon transport to the periplasm. The CRM197 is extracted byosmotic shock before being purified. The purification process is similarto the one disclosed in WO2006/100108 except that an additionalchromatographic step (Phenyl Sepharose) was added between theQ-Sepharose-XL and hydroxyapatite steps and the last chromatographicstep on the octyl-Sepharose 4FF was removed.

Preparation of Conjugates

It is well known in the art how to make purified pneumococcalpolysaccharides. For the purposes of these examples the polysaccharideswere made essentially as described in EP072513 or by closely-relatedmethods. Before conjugation the polysaccharides may be sized bymicrofluidisation as described below.

The activation and coupling conditions are specific for eachpolysaccharide. These are given in Table 1. Sized polysaccharide (exceptfor 6B and 23F) was dissolved in NaCl 2M, NaCl 0.2M or in water forinjection (WFI). The optimal polysaccharide concentration was evaluatedfor all the serotypes. All serotypes except serotype 18C were conjugateddirectly to the carrier protein as detailed below.

From a 100 mg/ml stock solution in acetonitrile or acetonitrile/water(50%/50%) solution, CDAP (1-cyano-4-dimethylaminopyridiniumtetrafluoroborate) (CDAP/PS ratio 0.5-1.5 mg/mg PS) was added to thepolysaccharide solution. 1.5 minute later, 0.2M-0.3M NaOH was added toobtain the specific activation pH. The activation of the polysaccharidewas performed at this pH for 3 minutes at 25° C. Purified protein(protein D, CRM197 or DT) (the quantity depends on the initialPS/carrier protein ratio) was added to the activated polysaccharide andthe coupling reaction was performed at the specific pH for up to 2 hour(depending upon serotype) under pH regulation. In order to quenchun-reacted cyanate ester groups, a 2M glycine solution was then added tothe mixture. The pH was adjusted to the quenching pH (pH 9.0). Thesolution was stirred for 30 minutes at 25° C. and thenincubatedovernight at 2-8° C. with continuous slow stirring.

Preparation of 18C:

18C was linked to the carrier protein via a linker—Adipic aciddihydrazide (ADH) Polysaccharide serotype 18C was microfluidized beforeconjugation.

Derivatization of tetanus toxoid with EDAC(2-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride)

For derivatization of the tetanus toxoid, purified TT was diluted at 25mg/ml in 0.2M NaCl and the ADH spacer was added in order to reach afinal concentration of 0.2M. When the dissolution of the spacer wascomplete, the pH was adjusted to 6.2. EDAC(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) was then added to reacha final concentration of 0.02M and the mixture was stirred for 1 hourunder pH regulation. The reaction of condensation was stopped byincreasing pH up to 9.0 for at least 30 minutes at 25° C.

Derivatized TT was then diafiltrated (10 kDa CO membrane) in order toremove residual ADH and EDAC reagent.

TT_(AH) (Tetanus toxoid conjugated to an ADH linker) bulk was finallysterile filtered until coupling step and stored at −70° C.

Chemical Coupling of TT_(AH) to PS 18C

Details of the conjugation parameters can be found in Table 1.

2 grams of microfluidized PS were diluted at the defined concentrationin water and adjusted to 2M NaCl by NaCl powder addition.

CDAP solution (100 mg/ml freshly prepared in 50/50 v/v acetonitrile/WFI)was added to reach the appropriate CDAP/PS ratio.

The pH was raised up to the activation pH 9.0 by the addition of 0.3MNaOH and was stabilised at this pH until addition of TT_(AH).

After 3 minutes, derivatized TT_(AH) (20 mg/ml in 0.2 M NaCl) was addedto reach a ratio TT_(AH)/PS of 2; the pH was regulated to the couplingpH 9.0. The solution was left one hour under pH regulation.

For quenching, a 2M glycine solution, was added to the mixturePS/TT_(AH)/CDAP.

The pH was adjusted to the quenching pH (pH 9.0).

The solution was stirred for 30 min at 25° C., and then overnight at2-8° C. with continuous slow stirring.

Specific Activation/Coupling/Quenching Conditions of S. PneumoniaeCapsular Saccharide-Protein D/TT/DT/PhtD/Ply Conjugates

Where “μfluid” appears in a row header, it indicates that the saccharidewas sized by microfluidisation before conjugation. Sizes of saccharidesfollowing microfluidisation are given in table 2.

TABLE 1 Specific activation/coupling/quenching conditions of S.pneumoniae capsular saccharide-Protein D/TT/DT/CRM197 conjugates 1 4 56A 7F Serotype μfluid μfluid mfluid mfluid 6B μfluid PS 2.27 2.37 7.1 105.0 5.0 conc. (mg/ml) PS WFI WFI WFI NaCl 2M NaCl 2M NaCl 2M dissolutionCarrier 10.0  10.0  5.0 10 5.0 10.0  conc. (mg/ml) PD PD PD CRM197 PD PDInitial 1.65/1 1.60/1 1/1 1/1 1.1/1 1.2/1 PROT/PS Ratio (w/w) CDAP conc.0.55 0.55  0.79   1.0  0.83  0.75 (mg/mg PS) pH_(a) = pH_(c) = pH_(q)9.0/9.0/9.0 9.5/9.5/9.0 9.0/9.0/9.0 9.5/9.5/9.0 9.5/9.5/9.0 9.5/9.5/9.09V 14 18C 19A 19F Serotype μfluid μfluid μfluid μfluid μfluid 23F PS 5.05.0  4.5 15.0 9.0 2.38 conc. (mg/ml) PS NaCl 2M NaCl 2M NaCl 2M NaCl 2MNaCl 2M NaCl 2M dissolution Carrier 10.0  10.0  20.0 15.0 20.0  5.0 protein (TT) (CRM197) (DT) conc. (mg/ml) Initial carrier 1.2/1 1.2/1 2/11.5/1 1.5/1 1/1 protein/PS Ratio (w/w) CDAP conc.  0.50  0.75  0.75  1.51.5 0.79 (mg/mg PS) pH_(a) = pH_(c) = pH_(q) 9.5/9.5/9.0 9.5/9.5/9.09.0/9.0/9.0 9.0/9.0/9.0 9.0/9.0/9.0 9.5/9.5/9.0 Note: pH_(a,c,q)corresponds to the pH for activation, coupling and quenching,respectivelyPurification of the Conjugates:

The conjugates were purified by gel filtration using a Sephacryl S400HRgel filtration column equilibrated with 0.15M NaCl (except S500HR wasused as buffer for 18C and 20 mM acetate containing 1.15MNaCl pH6.2 wasused for 19A) to remove small molecules (including DMAP) andunconjugated saccharide and protein. Based on the different molecularsizes of the reaction components, PS-PD, PS-TT, PS-CRM197 or PS-DTconjugates are eluted first, followed by free PS, then by free proteincarrier and finally DMAP and other salts (NaCl, glycine). Fractionscontaining conjugates are detected by UV_(280 nm). Fractions are pooledaccording to their Kd, sterile filtered (0.22 μm) and stored at +2-8° C.The PS/Protein ratios in the conjugate preparations were determined.

Formulation of the Vaccines

The 10V vaccine contains S. pneumoniae capsular saccharide serotype 1,4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F conjugates adsorbed ontoaluminium phosphate together at a human dose of 1, 3, 1, 1, 1, 1, 1, 3,3, 1 μg (the saccharides were individually adsorbed to aluminiumphosphate, they were then mixed together and the level of aluminiumphosphate adjusted to 500 μg).

The 12V vaccine was made in the same way as the 10V vaccine withadditional serotypes 19A and 6A conjugates at doses of 2 μg adsorbedonto aluminium phosphate added.

The 12V+ proteins vaccine was made in the same way as the 12V vaccineexcept proteins PhtD, dPly and PE-PilA were added. The PE-PilA wasproduced as described herein. The 12 conjugates and the proteins weremixed together using the dosages described for 12V above and theproteins at dosages of 30 μg each (note that this refers to 30 μg ofPE-PilA, not 30 μg of PE and 30 μg of PilA).

Example 22 Comparison of the Immunogenicity of 10V, 12V and 12V+Proteins Vaccines in Mice

Description of the Anti-Pneumococcal Polysaccharide PS (Polysaccharide)ELISA

Microplates were coated for 2 hours at 37° C. with capsularpolysaccharide (CPS) (100 μl per well of 2.5 μg/ml of PS1 and PS3, 5μg/ml of PS4, 5, 6A, 6B, 7F, 9V or 14; 10 μg/ml of PS19A and 23F or 40μg/ml of PS18C and PS19F in PBS). The plates were washed three timeswith NaCl 150 mM (0.9%)-Polysorbate 20 0.05%. Sera was diluted (1/2 for6A and 6B and 1/10 for the other serotypes) in PBS-Polysorbate20 0.05%containing CPS (1 mg CPS/ml of non diluted serum except or 6A and 6Bwhich was at 2.5 mg/ml) V/V and incubated for 1 hour at 37° C. in orderto neutralize antibodies directed to the CPS. The sera from miceimmunised as described in the section entitled ‘immunogenicity of threevaccine formulations in mice’ or a reference (an internal referencecalibrated with Chrompure mouse IgG) was added to the microwells andserially diluted 100 μl (two-fold dilution step) in PBS-Polysorbate200.05%. The plates were incubated under agitation for 30 minutes at roomtemperature. The plates were washed as above and anti-mouse IgGantibodies conjugated to peroxidase (100 μl per well) was added, theplates were incubated for 30 minutes at room temperature with shaking.After washing, the substrate (4 mg of OPDA (ortho phenylen-diamine) in10 ml of citrate 0.1M pH 4.5-4.6 and 5 μl of H₂O₂) was added to eachwell (100 μl) and the plates incubated for 15 minutes in the dark. Thereaction was stopped by addition of HCl 1N (50 μl). The absorbance wasread at 490 nm or 620 nm for the reference using a spectrophotometer.The color developed is directly proportional to the amount of antibodypresent in the serum.

Description of the ELISA to Measure PD, PE and PilA Antibodies

Plates were coated overnight at 4° C. with 100 μl per well of 2 μg/ml ofPD (1 mg/ml), 2 μg/ml of PE (1500 μg/ml), 2 μg/ml of PilA (3660 μg/ml)in carbonate buffer pH 9.6. The plates were washed four times with NaCl0.9% Polysorbate 20 0.05%. For PE and PilA ELISA, the plates weresaturated for 30 min at room temperature (with shaking) with PBS-BSA1%.After washing, sera from mice immunised as described in the sectionentitled ‘immunogenicity of three vaccine formulations in mice’ or areference (an internal reference calibrated with Chrompure mouse IgG)was added to microwells and serially diluted 100 μl (two-fold dilutionstep) in PBS Polysorbate 20 0.05% (for the PD assay) and PBS Polysorbate20 0.05% BSA 0.1% (for the PE and PilA assay). The plates were thenincubated for 30 min at room temperature with agitation. After washing,the plates were incubated with an anti-mouse IgG antibody conjugated toperoxidaseperoxidase (100 μl per well) at room temperature for 30minutes with shaking. The plates were then washed as above and thesubstrate conjugate (4 mg of OPDA (ortho phenylen-diamine) in 10 ml ofcitrate 0.1M pH 4.5-4.6 and 5 μl of H₂O₂) as added to each well (100 μl)for 15 min in darkness. The reaction was stopped by addition of HCl 1N50 μl and the absorbance was read at 490 nm (620 nm for the reference).

Description of the ELISAto Measure PhtD and dPly Antibodies

Plates were coated for 2 hours at 37° C. with 100 μl per well of 1 μg/mlof PhtD (1021 μg/ml) or 4 μg/ml of Ply (367 μg/ml). The plates were thenwashed three times with NaCl 0.09% Polysorbate 0.05%. After washing,sera from mice immunised as described in the section entitled‘immunogenicity of three vaccine formulations in mice’ or a reference(an internal reference calibrated with Chrompure mouse IgG) (was addedto microwells and serially diluted 100 μl (two-fold dilution step) inPBS Polysorbate 20 0.05%. The plates were incubated for 30 min at roomtemperature with agitation. After washing, the plates were incubatedwith an anti-mouse IgG antibody conjugated to peroxidase (100 μl perwell) at room temperature for 30 minutes with shaking. The plates werewashed as above and the substrate conjugate (4 mg of OPDA in 10 ml ofcitrate 0.1M ph 4.5 and 5 μl of H₂O₂) was added to each well (100 μl)for 15 min in darkness. The reaction was stopped by addition of HCl 1N50 μl and the absorbance was read at 490 nm (620 nm for the referencefilter).

Description of Opsonophagocytosis Assay (OPA)

Serum samples were heated for 45 min at 56° C. to inactivate anyremaining endogenous complement. Twenty-five microliters aliquots ofeach 1:2 diluted serum sample were two-fold serially diluted in 25 μlOPA buffer (HBSS (Hank's Balanced Salt Solution)—14.4% inactivated FCS(Foetal Calf Serum)) per well of a 96-well round bottom microtitreplate. Subsequently, 25 μl of a mixture of activated HL-60 cells (1×107cells/ml), freshly thawed pneumococcal working seed and freshly thawedbaby rabbit complement in an e.g. 4/2/1 ratio (v/v/v) (except forserotypes 1, 6B and 6A for which the ratio was 4/2/2) were added to thediluted sera to yield a final volume of 50 μl. The assay plate wasincubated for 2 h at 37° C. with orbital shaking (210 rpm) to promotethe phagocytic process. The reaction was stopped by laying themicroplate on ice for at least 1 min, the plate is kept on ice untiluse. A 20 μl aliquot of each well of the plate was then transferred intothe corresponding well of a 96-well flat bottom microplate and 50 μl ofTodd-Hewitt Broth-0.9% agar was added to each well. After overnightincubation at 37° C. and 5% CO₂, pneumococcal colonies which appeared inthe agar were counted using an automated image analysis system (KS 400,Zeiss, Oberkochen, Germany). Eight wells without serum sample were usedas bacterial controls to determine the number of pneumococci per well.The mean number of CFU of the control wells was determined and used forthe calculation of the killing activity for each serum sample. The OPAtitre for the serum samples was determined by the reciprocal dilution ofserum able to facilitate 50% killing of the pneumococci. Theopsonophagocytic titre was calculated by using a 4-parameter curve fitanalysis.

Immunogenicity of Three Vaccine Formulations in Mice.

2 groups of 27 female Balb/c mice distributed in 2 experiments wereimmunized by intramuscular (IM) injections on days 0, 14 and 28 with1/10 human dose of different formulation including Proteins alone (PhtD,dPly and PEPilA), Prevnar 13™ (a commercially available Streptococcalvaccine—results not presented) 10V, 12V (DSP2A017) and 12V+ proteins(DSP2A012) GMP lots. Mice received in a different leg (mimickingco-administration at different sites in infants in clinical trials)1/10^(th) human dose of Infanrix Hexa™ (a vaccine comprising diphtheriatoxoid, tetanus toxoid, pertussis toxoid, filamentous haemmagglutinin,pertactin, hepatitis B surface antigen, inactivated polio virus types 1,2 and 3 and Haemophilus influenzae b saccharide (PRP)).

Anti-IgG levels and opsonophagocytosis titers were determinedrespectively in individual and pooled sera collected at day 42.

The potential of the 12V+ proteins vaccine to induce IgG antibody titersand opsonic activity was evaluated and compared to that of the 12V and10V vaccines.

Sera from mice injected with the different formulations were tested inELISA (as described above) against the polysaccharide serotypes andproteins and in OPA against the 12 polysaccharide serotypes.

The 12V+ proteins vaccine induced similar response to the 12V for mostserotypes.

The results in FIG. 31 demonstrated that there was no stasticaldifference between the PE, PilA, PhtD, Ply and PD antibody responsesmeasured for the 12V+ formulation and a formulation that did notcomprises the S. pneumoniae saccharide conjugates.

Example 23 Comparison of the Immunogenicity of 10V, 12V and 12V+ ProteinVaccines in Guinea Pigs

Description of ELISA Anti-Pneumococcal Polyssacharide PS

Microplates were coated for 2 hours at 37° C. with 100 μl per well of2.5 μg/ml of PS1, 5 μg/ml of PS4, 5, 6A, 6B, 7F, 9V or 14; 10 μg/ml ofPS19A and 23F, 40 μg/ml of PS18C and PS19F or Affinipure Goatanti-guinea pig IgG (2.4 mg/ml) diluted to 2 μg/ml for the referencewells in PBS. The plates were washed three times with NaCl 150 mM(0.9%)-Polysorbate20 0.05%. Pooled serum from each group was diluted (½for P δ 6A and 6B and 1/10 for all the other serotypes) inPBS-Polysorbate20 0.05% containing CPS (1 mg CPS/ml of non diluted serumexcept for 6A and 6B at 2.5 mg/ml) V/V and incubated for 1 hour at 37°C. in order to neutralize antibodies directed to CPS. The serum fromguinea pigs immunised as described in the section entitled‘immunogenicity of three vaccine formulations in guinea pigs’ was addedto the microwells and serially diluted 100 μl (two-fold dilution step)in PBS-Polysorbate20 0.05% or a reference (Chrompure guinea pig IgG (11mg/ml) diluted to 0.25 μg/ml in PBS-polysorbate20 0.05%) was added. Theplates were incubated under agitation for 30 minutes at roomtemperature. The plates were washed as above and anti-guinea pig IgGantibodies conjugated to peroxidase (100 μl per well) were added and theplates incubated for 30 minutes at room temperature with shaking. Afterwashing, the substrate (4 mg of OPDA in 10 ml of citrate 0.1M pH 4.5-4.6and 5 μl of H₂O₂) was added to each well (100 μl) and incubated for 15minutes in the dark. The reaction was stopped by addition of HCl 1N.Absorbance was read at 490 nm (620 nm for the reference) using aspectrophotometer. The color developed is directly proportional to theamount of antibody present in the serum.

Description of the ELISA to Measure Anti PD, PE, and PilA, Antibodies

Plates were coated for 2 hours at 37° C. with 100 μl per well of 2 μg/mlof PD (1 mg/ml), 2 μg/ml of PE (1500 μg/ml), or 2 μg/ml of PilA (3660μg/ml) in carbonate buffer pH 9.6 in PBS or Affinipure Goat anti-guineapig IgG (2.4 mg/ml) diluted to 2 μg/m for the reference wells in PBS.The plates were washed 4 times with NaCl 0.9% Polysorbate 20 0.05%. ForPE and PilA ELISAs (this step was not carried out for the PD and PlyELISAs), the plates were saturated 30 min at room temperature withPBS-BSA1%. After washing, sera from guinea pigs immunised as describedin the section entitled ‘immunogenicity of three formulations in guineapigs’ or a reference serum sample (an internal reference calibrated withChrompure guinea pig IgG) was added to microwells and serially diluted100 μl (two-fold dilution step) in PBS Polysorbate 20 0.05% (for the PDELISA) and PBS Polysorbate 20 0.05% BSA 0.1% for the PE and PilA ELISAs.The plates were incubated for 30 min at room temperature. After washing,the plates were incubated with an anti-guinea pig IgG antibodyconjugated to peroxydase (100 μl per well) at room temperature for 30minutes with shaking. Plates were washed as above and the substrateconjugate (4 mg of OPDA in 10 ml of citrate 0.1M pH 4.5-4.6 and 5 μl ofH₂O₂) was added to each well (100 μl) for 15 min in darkness. Thereaction was stopped by addition of HCl 1N 50 μl and the absorbance isread at 490 nm (620 nm for the reference filter).

Description of the ELISA to Measure PhtD and dPly Antibodies

Plates were coated for 2 hours at 37° C. with 100 μl per well of 1 μg/mlof PhtD (1021 μg/ml) or 2 μg/ml Ply (376 μg/ml) in PBS. The plates werethen washed 4 times with NaCl 0.9% Polysorbate 20 0.05%. After washing,sera from guinea pigs immunised as described in the section entitled‘immunogenicity of three vaccine formulations in guinea pigs’ or areference (an internal reference calibrated with Chrompure guinea pigIgG) was added to microwells and serially diluted 100 μl (two-folddilution step) in PBS Polysorbate 20 0.05%. The plates were incubatedfor 30 min at room temperature with agitation. After washing, the plateswere incubated with an anti-guinea pig IgG antibody conjugated toperoxydase (100 μl per well) at room temperature for 30 minutes withshaking. Plates were washed as above and the substrate conjugate (4 mgof OPDA in 10 ml of citrate 0.1M pH 4.5-4.6 and 5 μl of H₂O₂) was addedto each well (100 μl) for 15 min in darkness. The reaction was stoppedby addition of HCl 1N 50 μl and the absorbance was read at 490 nm (620nm for the reference filter).

Opsonophagocytosis Assay

Serum samples were heated for 45 min at 56° C. to inactivate anyremaining endogenous complement. Twenty-five microliters aliquots ofeach 1:2 diluted serum sample was two-fold serially diluted in 25 μl OPAbuffer (HBSS (Hank's Balanced Salt Solution)—14.4% inactivated FCS(foetal calf serum)) per well of a 96-well round bottom microtitreplate. Subsequently, 25 μl of a mixture of activated HL-60 cells (1×107cells/ml), freshly thawed pneumococcal working seed and freshly thawedbaby rabbit complement in an e.g. 4/2/1 ratio (v/v/v) (except forserotypes 1,6B and 6A for which the ratio was 4/2/2) was added to thediluted sera to yield a final volume of 50 μl. The assay plate wasincubated for 2 h at 37° C. with orbital shaking (210 rpm) to promotethe phagocytic process. The reaction was stopped by laying themicroplate on ice for at least 1 min (the plate should be kept on iceuntil further use. A 20 μl aliquot of each well of the plate was thentransferred into the corresponding well of a 96-well flat bottommicroplate and 50 μl of Todd-Hewitt Broth-0.9% agar was added to eachwell. After overnight incubation at 37° C. and 5% CO2, pneumococcalcolonies appearing in the agar were counted using an automated imageanalysis system (KS 400, Zeiss, Oberkochen, Germany). Eight wellswithout serum sample were used as bacterial controls to determine thenumber of pneumococci per well. The mean number of CFU of the controlwells was determined and used for the calculation of the killingactivity for each serum sample. The OPA titre for the serum samples wasdetermined by the reciprocal dilution of serum able to facilitate 50%killing of the pneumococci. The opsonophagocytic titre was calculated byusing a 4-parameter curve fit analysis.

Immunogenicity of Three Formulations in Guinea Pigs

2 groups of 17 guinea pigs distributed in 2 experiments were immunizedby intramuscular (IM) injections on days 0, 14 and 28 with ¼ human doseof different formulation including Proteins alone (PhtD, dPly andPEPilA), Prevnar 13(™ a commercially available Streptococcalvaccine—results not presented) 10V, 12V (DSP2A017) and 12V+ proteins(DSP2A012) GMP lots. Guinea pigs received in a different leg (mimickingco-administration at different sites in infants in clinical trials) ¼ ofhuman dose of Infanrix Hexa((™ a vaccine comprising diphtheria toxoid,tetanus toxoid, pertussis toxoid, filamentous haemmagglutinin,pertactin, hepatitis B surface antigen, inactivated polio virus types 1,2 and 3 and Haemophilus influenzae b saccharide (PRP). Anti-IgG levelsand opsonophagocytosis titers were determined respectively in individualand pooled sera collected at days 42.

The IgG antibody titers and opsonic activity was evaluated and comparedbetween the 12V+ proteins, 12V and 10V vaccines.

Sera from guinea pigs injected with the different formulations weretested in ELISA against the polysaccharide serotypes and proteins and inOPA against the 12 serotypes in the formulation. The 12V+ proteinsinduced similar responses to the 12V formulation.

The results in FIG. 34 demonstrated that there is no negative impact onthe immunogenicity of the 12 valent conjugates when they are combinedwith PEPilA.

The results obtained with the 12V+ proteins GMP formulation demonstratedimmunogenicity of the 10V polysaccharides as well as the proteins andsupport the clinical evaluation of this formulation.

We claim:
 1. An immunogenic composition comprising: (i) 1 or moreStreptococcus pneumoniae capsular saccharide conjugates; and (ii) afusion protein component comprising (a) an immunogenic fragment ofProtein E wherein the amino acid sequence of the immunogenic fragment ofProtein E has a sequence that is SEQ ID NO. 124 or SEQ ID NO. 125, and(b) an immunogenic fragment of PilA from Haemophilus influenzae, whereinthe amino acid sequence of the immunogenic fragment of PilA has at least95%, identity, over the entire length, to SEQ ID NO. 127, wherein theimmunogenic fragment of protein E is covalently linked to immunogenicfragment of PilA to form the fusion protein.
 2. The immunogeniccomposition of claim 1 wherein the immunogenic fragment of Protein E isa polypeptide having an amino acid sequence of SEQ ID NO.
 124. 3. Theimmunogenic composition of claim 1 wherein the immunogenic fragment ofProtein E is a polypeptide having the amino acid sequence of SEQ ID NO.125.
 4. The immunogenic composition of claim 1 wherein the immunogenicfragment of PilA is a polypeptide comprising a sequence having at least98% identity, over the entire length, to SEQ ID NO.
 127. 5. Theimmunogenic composition of claim 1 wherein the immunogenic fragment ofPilA is a polypeptide having an amino acid sequence that is SEQ ID NO.127.
 6. The immunogenic composition of claim 1 wherein the fusionprotein is at least 95%, identical to SEQ ID NO.
 194. 7. The immunogeniccomposition of claim 1 wherein the 1 or more Streptococcus pneumoniaecapsular saccharide conjugates comprises a conjugated saccharideselected from the group consisting of a Streptococcus pneumoniaeserotype 1, serotype 4, serotype 5, serotype 6B, serotype 9V, a serotype14, a serotype 18C, a serotype 19F and a serotype 23F capsularsaccharide conjugate.
 8. The immunogenic composition of claim 1 or claim7 wherein the 1 or more Streptococcus pneumoniae capsular saccharideconjugates comprises a conjugated saccharide selected from the groupconsisting of a conjugated serotype 1 saccharide, a conjugated serotype5 saccharide and a conjugated serotype 7F saccharide.
 9. The immunogeniccomposition of claim 1 or claim 7 wherein the 1 or more Streptococcuspneumoniae capsular saccharide conjugates are conjugated to a carrierprotein independently selected from the group consisting of tetanustoxoid (TT), fragment C of TT, diphtheria toxoid, CRM197 (cross reactingmaterial 197), Pneumolysin, protein D (from H. influenzae), PhtD (PolyHistidine Triad D), PhtDE (a fusion between Pneumococal Histidine TriadD and E) and N19.
 10. The immunogenic composition of claim 1 or claim 7wherein the Streptococcus pneumoniae capsular saccharide conjugates areall independently conjugated to CRM197.
 11. The immunogenic compositionof claim 1 or claim 7 wherein the 1 or more Streptococcus pneumoniaecapsular saccharide conjugates comprises a serotype 1 saccharideconjugated to protein D, a serotype 4 saccharide conjugated to proteinD, a serotype 5 saccharide conjugated to protein D, a serotype 6Bsaccharide conjugated to protein D, a serotype 7F saccharide conjugatedto protein D, a serotype 9V saccharide conjugated to protein D, aserotype 14 saccharide conjugated to protein D, a serotype 23Fsaccharide conjugated to protein D, a serotype 18C saccharide conjugatedto tetanus toxoid and a serotype 19F saccharide conjugated to diphtheriatoxoid.
 12. The immunogenic composition of claim 1 wherein theimmunogenic composition further comprises at least one unconjugated orconjugated Streptococcus pneumoniae protein.
 13. The immunogeniccomposition of claim 12 wherein the at least one unconjugated orconjugated Streptococcus pneumoniae protein is a PhtD protein.
 14. Theimmunogenic composition of claim 12 wherein the at least oneunconjugated or conjugated Streptococcus pneumoniae protein isdetoxified pneumolysin (dPly).
 15. A vaccine comprising the immunogeniccomposition of claim 1 or claim 7 and a pharmaceutically acceptableexcipient.
 16. A method of immunizing a subject against diseases causedby Streptococcus pneumoniae infection comprising administering to thesubject a therapeutically effective dose of the immunogenic compositionof claim 1 or claim 7 or the vaccine of claim
 15. 17. A method ofimmunizing a subject against diseases caused by Haemophilus influenzaeinfection comprising administering to the subject a therapeuticallyeffective dose of the immunogenic composition of claim 1 or claim 7 orthe vaccine of claim
 15. 18. An immunogenic composition comprising afusion protein wherein the amino acid sequence of the fusion protein isSEQ ID NO.
 219. 19. An immunogenic composition comprising a fusionprotein wherein the amino acid sequence of the fusion protein is SEQ IDNO. 177.